Note: Descriptions are shown in the official language in which they were submitted.
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DUAL MHC-TARGETING T CELL ENGAGER
RELATED APPLICATIONS
[001] This application claims the benefit of U.S. Provisional Application
Serial No.
63/289,380, filed December 14, 2021, U.S. Provisional Application Serial No.
63/317,256, filed
March 7, 2022, and U.S. Provisional Application Serial No. 63/328,417, filed
April 7, 2022, the
entire disclosures of which are hereby incorporated herein by reference.
HELD 011"tHE DISCLOSURE
[002] The present disclosure relates to highly potent T-cell engager antibody
formats that
bind to tumor peptide-M_HC (pMHC) complexes with high specificity and further
comprise a CD3
targeting moiety in a Fab format. Such pMHC T-cell engagers rely on bivalent
pMHC binding
and a monovalent CD3 binding in different affinities for cytokine release
tunning. The bivalent
targeting of pMHCs on cancer cells provides efficient T-cell mediated cancer
cell killing despite
very low levels of pMHC on the cell surface
BACKGROUND
[003] Peptide-MEC complexes (pMHCs) derived from intracellular tumor
associated
antigens (TAAs) represent a large repertoire of novel targets for
immunotherapy. pMTICs are
present on the surface of virtually all nucleated cells and are constantly
surveilled by T-cells.
Upon pMFIC binding by T-cell receptors (TCRs), infected and/or malignantly
transformed cells
are recognized and eliminated. Thus, intracellular tumor associated proteins
presented as peptides
on MI-IC class I molecules are attractive targets for immunotherapeutic
approaches with
promising data already emerging from clinical trials. pMHCs have been
traditionally targeted by
TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to
an anti-CD3
fragment. However, naturally occurring cancer reactive TCRs typically exhibit
binding affinities
between 0.1-500 ILIM for their pMHC targets. Therefore, they need substantial
engineering efforts
to endow them with the necessary binding affinity and biophysical properties
to be developed as
drugs which may compromise the required specificity to the pMFIC target.
Conversely,
developing high-affinity soluble antibody molecules with high specificity to
pMHCs derived from
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the intracellular tumor associated antigens, addresses the challenging low
affinity of TCRs which
require significant affinity enhancements.
SUMMARY
[004] The present disclosure relates to antigen binding proteins comprising a
Fab domain
which specifically binds to a cell surface protein of an immune cell, the Fab
domain comprising
a heavy chain and a light chain; at least a first pMHC binding domain operably
linked to the heavy
chain, wherein the first pMHC binding domain binds to first target peptide-MHC
(pMHC)
complex; and c) at least a second pMHC binding domain operably linked to the
light chain,
wherein the second pMHC binding domain binds to a second pMHC complex.
Bivalent targeting
of pMHCs with the bispecific antigen binding proteins of the invention results
in increased cancer
cell killing compared to their monovalent bispecific counterparts, while the
overall specificity
against cells bearing the same BLA allele but not expressing the target
protein is not substantially
affected.
[005] The antigen binding proteins of the invention lack an Fe domain. The
antigen
binding proteins of the disclosure therefore are not recognized through Fe-
receptors on effector
cells, such as the Fe-receptor FcyRIII on macrophages and activated
neutrophils, or inhibiting
receptors such as FcyRIIb, and on FcyRIIa complexes on non-cytotoxic cells
such as platelets and
B-cells. For bispecific T-cell engagers, Fe-mediated immune functions are
unwanted to avoid
antigen-independent cytokine release syndrome (CRS) due to crosslinking of CD3
and Fcy
receptors followed by nonspecific activation of immune cells. Rather, the Fab
domain of the
antigen binding protein serves as a specific heterodimerization scaffold to
which the additional
pMHC binding domains are linked. The natural and efficient heterodimerization
properties of the
heavy chain (Fd fragment) and light chain (L) of a Fab fragment makes the Fab
fragment a useful
scaffold. Additional binding domains may be in several different formats,
including, but not
limited to, another Fab domain, a scFv, or an sdAb. Moreover, in certain
contexts, an Fc-
containing antigen binding protein may be disadvantageous due to increased
half-life. An
extended half-life may lead to increased toxicity from, among other things,
excess cytokine
release from immune cells. The extended half-life may also promote T cell
exhaustion. The
antigen binding proteins of the disclosure lacking an Fe domain may possess
reduced cytotoxicity
in part due to a reduce half-life relative to an Fe-containing antigen binding
protein.
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[006] In one aspect, the disclosure provides an antigen binding protein
comprising: a) a
single Fab domain which specifically binds to a cell surface protein of an
immune cell, the Fab
domain comprising a heavy chain and a light chain; b) at least a first pMHC
binding domain
operably linked to the heavy chain, wherein the first pMHC binding domain
binds to first target
peptide-M_HC (pMHC) complex, and c) at least a second pMHC binding domain
operably linked
to the light chain, wherein the second pMHC binding domain binds to a second
pMHC complex,
wherein antigen binding protein does not comprise an Fe domain.
[007] In certain embodiments, the Fab domain heavy chain comprises a CHI
domain and
a VH domain, and at least 5 amino acids of an antibody hinge region. In
certain embodiments
thereof, the Fab domain heavy chain comprises at most 10 amino acids of an
antibody hinge region
at the C-terminus of the CH1 domain. In certain embodiments, the Fab domain
heavy chain
comprises 5-10 amino acids of an antibody hinge region at the C-terminus of
the CH1 domain. In
certain embodiments, said at least 5 amino acids or said at most 10 amino
acids of an antibody
hinge region comprise the sequence EPKSC (SEQ ID NO 87) Additionally, the at
least 5 amino
acids, respectively the at most 10 amino acids of an antibody hinge region,
may be followed by a
GGGGS (SEQ ID NO.: 88) linker.
[008] In certain embodiments, the Fab domain light chain comprises a CL domain
and a
VL domain. The CL domain may be followed by a linker, such as GGGGS (SEQ ID
NO. :88).
[009] In certain embodiments, the first target pMHC complex and the second
target
pMHC complex are the same. In certain embodiments, the first target pMHC
complex and the
second target pMHC complex are different.
[010] In certain embodiments, the first pMHC binding domain is operably linked
to the
C-terminus of the heavy chain or the N-terminus of the heavy chain. In certain
embodiments, the
second pMHC binding domain is operably linked to the C-terminus of the heavy
chain or the N-
terminus of the heavy chain.
[011] In certain embodiments, the first pMTIC binding domain is operably
linked to the
C-terminus of the light chain or the N-terminus of the light chain. In certain
embodiments, the
second plVIFIC binding domain is operably linked to the C-terminus of the
light chain or the N-
terminus of the light chain.
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[012] In certain embodiments, the pMHC binding domain is a scFv or an sdAb. As
described elsewhere herein, the pMHC binding domain may also be any one of a
scFab, a diabody
or a Fab.
[013] In certain embodiments, the antigen binding protein comprises. 1) a
first
binding scFv linked to the C-terminus of the Fab domain heavy chain and a
second pMHC binding
scFv linked to the C-terminus of the Fab domain light chain; 2) a first pMHC
binding scFv linked
to the N-terminus of the Fab domain heavy chain and a second pMHC binding scFv
linked to the
N-terminus of the Fab domain light chain; 3) a first pMHC binding scFv linked
to the N-terminus
of the Fab domain heavy chain and a second pMHC binding scFv linked to the C-
terminus of the
Fab domain light chain; 4) a first pMHC binding scFv linked to the C-terminus
of the Fab domain
heavy chain and a second pMHC binding scFv linked to the N-terminus of the Fab
domain light
chain; 5) a first pMEIC binding sdAb linked to the N-terminus of the Fab
domain heavy chain and
a second pMHC binding sdAb linked to the N-terminus of the Fab domain light
chain; 6) a first
pMHC binding sdAb linked to the C-terminus of the Fab domain heavy chain and a
second pMHC
binding sdAb linked to the C-terminus of the Fab domain light chain; 7) a
first pMHC binding
sdAb linked to the N-terminus of the Fab domain heavy chain and a second pMHC
binding sdAb
linked to the C-terminus of the Fab domain light chain; or 8) a first pMHC
binding sdAb linked
to the C-terminus of the Fab domain heavy chain and a second pMHC binding sdAb
linked to the
N-terminus of the Fab domain light chain.
[014] In certain embodiments, the first plVIEIC binding domain and/or the
second plVIHC
binding domain comprise a variable heavy chain having a polar amino acid at
position 11, 89
and/or 108, according to Kabat numbering.
[015] In certain embodiments, the Fab domain comprises a variable heavy chain
having
a non-polar amino acid at position 11, 89 and/or 108, according to Kabat
numbering.
[016] In certain embodiments, the variable heavy chain comprises: leucine (L)
or serine
(S) at amino acid position 11, according to Kabat numbering; valine (V),
serine (S), or threonine
(T) at amino acid position 89, according to Kabat numbering; and/or leucine
(L), serine (S), or
threonine (T) amino acid position 108, according to Kabat numbering.
[017] In certain embodiments, the polar amino acid is serine (S) and/or
threonine (T).
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[018] In certain embodiments, the variable heavy chain comprises serine (S) at
amino
acid position 11, serine (S) or threonine (T) at amino acid position 89, and
serine (S) or threonine
(T) at amino acid position 108, according to Kabat numbering.
[019] In certain embodiments, the variable heavy chain comprises serine (S) at
amino
acid position 11, serine (S) at amino acid position 89, and serine (S) at
amino acid position 108,
according to Kabat numbering.
[020] In certain embodiments, the Fab domain comprises a variable heavy chain
having
a serine (S) at position 113 deleted, according to Kabat numbering.
[021] In certain embodiments, the first pMHC binding domain and/or the second
pMTIC
binding domain comprise a variable heavy chain having a serine (S) at position
113 deleted,
according to Kabat numbering.
[022] In certain embodiments, the Fab domain comprises a variable heavy chain
having
a serine (S) at position 112 deleted and a serine (S) at position 113 deleted,
according to Kabat
numbering.
[023] In certain embodiments, the first pMHC binding domain and/or the second
pMHC
binding domain comprise a variable heavy chain having a serine (S) at position
112 deleted and a
serine (S) at position 113 deleted, according to Kabat numbering.
[024] In certain embodiments, the antigen binding protein comprises an Si 13A,
Si 13G,
or Si 13T substitution, according to Kabat numbering.
[025] In certain embodiments, the antigen binding protein comprises an Si 13A,
S113G,
or Si 13T substitution, and wherein S112 is deleted, according to Kabat
numbering.
[026] In certain embodiments, the antigen binding protein comprises an Si 12A,
Si 12G,
or Si 12T substitution, according to Kabat numbering.
[027] In certain embodiments, the antigen binding protein comprises an Si 12A,
Si 12G,
or 51 12T substitution, and wherein S113 is deleted, according to Kabat
numbering.
[028] In certain embodiments, the target pM_HC binding domain specifically
targets an
MEW restricted peptide derived of a tumor antigen or a viral antigen.
[029] In certain embodiments, the cell surface protein of an immune cell is
selected from
the group consisting of CD3, TCRa.õ TCRp, CD16, NKG2D, CD89, CD64, and CD32a
In certain
embodiments, the cell surface protein of an immune cell is CD3.
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[030] In certain embodiments, the immune cell is selected from the group
consisting of
a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell,
a neutrophil cell, a
monocyte, and a macrophage. In certain embodiments, the immune cell is a T
cell.
[031] In certain embodiments, the Fab domain specifically binds to CD3 with a
binding
affinity (KD) between about 1 nM to about 50 nM, optionally between about 20
nM to 50 nM, as
determined by SPR.
[032] In certain embodiments, the Fab domain specifically binds to CD3 with a
binding
affinity (KD) of about 1 nM, of about 10 nM, or of about 50 nM, as determined
by SPR.
[033] In certain embodiments, the first pMHC binding domain and/or the second
pMHC
binding domain bind the target pMHC complex with a binding affinity (KD) of
about 100 pM to
about 5 n1\4. In certain embodiments, the first pMHC binding domain and/or the
second pMHC
binding domain bind the target pMHC complex with a binding affinity (KD) of
about 500 pM to
about 5 nM, to about 10 nM, or to about 20 nM.
[034] In certain embodiments, the antigen binding protein comprises a
molecular weight
of about 75 kDa to about 100 kDa, or of about 75 kDa to about 105 kDa or 110
kDa.
[035] In certain embodiments, the antigen binding protein has increased serum
half-life
relative to an antigen binding protein with a molecular weight of less than
about 60 kDa.
[036] Thus, in one aspect, the disclosure provides an antigen binding protein
comprising:
a) a single Fab domain which specifically binds CD3 on a T cell, the Fab
domain comprising a
heavy chain and a light chain; b) at least a first pMHC binding domain
operably linked to the C-
temiinus of the heavy chain, wherein the first pl\TEIC binding domain binds to
first target peptide-
MHC (pMHC) complex; and c) at least a second pMHC binding domain operably
linked to the
C-terminus of the light chain, wherein the second plVIFIC binding domain binds
to a second p1\41FIC
complex, wherein antigen binding protein does not comprise an Fc domain.
[037] In one aspect, the disclosure provides a method of reducing nonspecific
T cell
activation of a T cell engaging multispecific antigen binding protein, wherein
the multispecific
antigen binding protein comprises a first binding domain specifically
targeting CD3 and a second
binding domain specifically targeting a tumor antigen, wherein the
multispecific antigen binding
protein comprises at least one variable heavy chain, the method comprising the
step of: a)
substituting a variable heavy chain amino acid at position 11, 89, and/or 108,
according to Kabat
numbering, with a polar amino acid.
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[038] In certain embodiments, the method further comprises the step of: b)
deleting a
serine (S) at position 113, according to Kabat numbering.
[039] In certain embodiments, the polar amino acid of step a) is serine (S)
and/or
threonine (T).
[040] In certain embodiments, the heavy chain amino acid is substituted with
serine (S)
at heavy chain amino acid position 11, serine (S) or threonine (T) at heavy
chain amino acid
position 89, and/or serine (S) or threonine (T) at heavy chain amino acid
position 108, according
to Kabat numbering.
[041] In certain embodiments, the heavy chain amino acid is substituted with
serine (S)
at heavy chain amino acid position 1 1 , serine (S) at heavy chain amino acid
position 89, and serine
(S) at heavy chain amino acid position 108, according to Kabat numbering.
[042] In certain embodiments, step b) further comprises the step of deleting a
serine (S)
at position 112, according to Kabat numbering.
[043] In certain embodiments, the method further comprises adding alanine (A),
glycine
(G) or threonine (T) at Kabat amino position 112 or 113.
[044] In certain embodiments, the method comprises adding alanine (A) at Kabat
amino
position 112 or 113.
[045] In certain embodiments, the substitutions and/or deletions are made in
the heavy
chain of the second binding domain.
[046] In certain embodiments, the multispecific antigen binding protein is
monovalent,
bivalent or multivalent.
[047] In certain embodiments, the antigen binding protein which may be used in
such
method is a Fab-sdAb, Fab-(sdAb)2, a Fab-scFy or a Fab-(scFv)2,
F(ab)2fragment, bis-scFv (or
tandem scFy or BiTE), DART, diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc-
scFab, IgG-scFv,
scFv-Fc, scFv-fc-scFv, Fv2-Fc, FynomAB, quadroma, CrossMab, DuoBody, triabody
and
tetrabody, or MATCH.
[048] In certain embodiments, the second binding domain specifically targets a
pMEIC.
In certain embodiments, the multispecific antigen binding protein further
comprises a third
binding domain specifically targeting a pMFIC. In certain embodiments, the
second binding
domain and the third binding domain specifically target the same pMHC or
different pMHC.
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[049] In certain embodiments, the antigen binding protein comprises one
binding domain
specifically targeting CD3 and one binding domain specifically targeting a
pMHC.
[050] In certain embodiments, the antigen binding protein comprises one
binding domain
specifically targeting CD3 and two binding domains specifically targeting a
pMHC.
[051] In certain embodiments, the two binding domains specifically targeting a
pMFIC
are the same. In some embodiments, the two pMHC binding domains comprise the
same set of
six CDR sequences. In some embodiments, the two plVIFIC binding domains
comprise the same
VL and VH sequences.
[052] Thus, in certain embodiments, the antigen binding protein is a Fab-
(scFv)2,
wherein the Fab targets CD3 and one or both scFy target a tumor antigen, in
particular a pMHC
complex, such as a MAGE-A4 derived peptide presenting HLA as outlined below.
The
substitutions and/or deletions described herein are made in the heavy chain of
the scFvs.
[053] In certain embodiments, the pMHC binding domain specifically targets a
MHC
restricted peptide derived of a tumor antigen or a viral antigen.
[054] In certain embodiments, the binding affinity (KD) for CD3 is between
about 1 nM
to about 50 nM, optionally between about 20 nM to 50 nM, as determined by SPR.
In certain
embodiments, the binding affinity (KD) for CD3 is of about 1 nM, of about 10
nM, or of about 50
nM, as determined by SPR. In certain embodiments, the binding affinity (KD)
for CD3 is of about
1 nM, of about 10 nM, or of about 50 nM, as determined by SPR.
[055] In certain embodiments, the binding affinity (KD) for the plVIEIC is of
about 100
pM to about 20 nM, such as about 500 pM to about 10 nM or about 500 pM to
about 5 nM or
about 500 pM to about 2 nM.
[056] In one aspect, the disclosure provides a multispecific antigen binding
protein
obtainable by the method described above.
[057] In one aspect, the disclosure provides an antigen binding protein
comprising at
least one first binding domain specific for CD3 and at least one second
binding domain specific
for a tumor antigen, each binding domain comprising at least one variable
heavy chain, wherein
at least one variable heavy chain comprises a polar amino acid at position 11,
89 and/or 108,
according to Kabat numbering.
[058] In certain embodiments, the variable heavy chain is of said second
binding domain.
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[059] In certain embodiments, the polar amino acid is serine (S) and/or
threonine (T).
[060] In certain embodiments, the variable heavy chain comprises serine (S) at
heavy
chain amino acid position 11, serine (S) or threonine (T) at heavy chain amino
acid position 89,
and serine (S) or threonine (T) at heavy chain amino acid position 108,
according to Kabat
numbering.
[061] In certain embodiments, the variable heavy chain comprises serine (S) at
heavy
chain amino acid position 11, serine (S) at heavy chain amino acid position
89, and serine (S) at
heavy chain amino acid position 108, according to Kabat numbering
[062] In certain embodiments, the variable heavy chain has a serine (S) at
position 113
deleted, according to Kabat numbering.
[063] In certain embodiments, the variable heavy chain has serine (S) at
position 112 and
113 deleted, according to Kabat numbering.
[064] In certain embodiments, the antigen binding protein comprises alanine
(A), glycinc
(G) or threonine (T) at position 112, according to Kabat numbering, in
particular alanine (A)
[065] In certain embodiments, the antigen binding protein comprises alanine
(A), glycine
(G) or threonine (T) at position 112, according to Kabat numbering, in
particular alanine (A)
[066] In certain embodiments, the tumor antigen is a pMHC.
[067] In certain embodiments, the pMHC binding domain specifically targets a
MEC
restricted peptide derived of a tumor antigen or a viral antigen.
[068] In certain embodiments, the antigen binding protein has an affinity (KD)
for CD3
of about 1 nM to about 50 nM, preferably between about 20 nM to 50 nM, as
determined by SPR.
In certain embodiments, the antigen binding protein has an affinity (KD) for
CD3 of about 1 nM,
of about 10 nM, or of about 50 nM, as determined by SPR.
[069] In certain embodiments, the first binding domain specific for CD3 is a
Fab
fragment.
[070] In certain embodiments, the antigen binding protein comprises two or
more pMHC
binding domains
[071] In certain embodiments, the pMHC binding domain is a scFy or an sdAb.
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[072] In certain embodiments, the antigen binding protein has an affinity (KD)
for the
pMEIC of about 100 pM to about 20 nM, such as about 500 pM to about 10 nM or
about 500 pM
to about 5 nM.
[073] In certain embodiments, the antigen binding protein is a Fab-sdAb, Fab-
(sdAb)2, a
Fab-scFv or a Fab-(scFv)2, F(a131)2fragment, bis-scFv (or tandem scFv or
BiTE), DART,
diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc-scFab, IgG-scFv, scFv-Fc, scFv-fc-
scFv, Fv2-Fc,
FynomAB, quadroma, CrossMab, DuoBody, triabody and tetrabody, or MATCH.
[074] In one aspect, the disclosure provides a method for killing a target
cell comprising
a major histocompatibility complex (1\41-1C) presenting a neoantigen, the
method comprising. a)
contacting a plurality of cells comprising immune cells and the target cell
with the antigen binding
protein described above, wherein said antigen binding protein specifically
binds to the pMHC on
the surface of the target cell and to CD3 on the surface of the immune cells;
b) forming a specific
binding complex through the antigen binding protein interactions with the
target cells and the
immune cells, thereby activating the immune cells; and c) killing the target
cell with the activated
immune cells
[075] In one aspect, the disclosure provides a composition comprising an
antigen binding
protein described herein.
[076] In one aspect, the disclosure provides a method of treating cancer
comprising the
step of administering the composition described above to a patient in need
thereof.
[077] In one aspect, the disclosure provides a nucleic acid encoding an
antigen binding
protein described herein.
[078] An expression vector comprising the nucleic acid described above.
[079] In one aspect, the disclosure provides a host cell population comprising
the
expression vector described above.
[080] In one aspect, the disclosure provides a kit comprising an antigen
binding protein
described herein.
[081] In one aspect, the disclosure provides a method of manufacturing an
antigen
binding protein as described herein, comprising the steps of: (i) cultivating
the host cell described
above under conditions allowing expression of the antigen binding protein
described above; (ii)
recovering the antigen binding protein or bispecific antigen binding protein,
and optionally (iii)
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further purifying and/or modifying and/or formulating the antigen binding
protein or bispecific
antigen binding protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[082] The foregoing and other features and advantages of the present invention
will be
more fully understood from the following detailed description of illustrative
embodiments taken
in conjunction with the accompanying drawings. The patent or application file
contains at least
one drawing executed in color. Copies of this patent or patent application
publication with color
drawing(s) will be provided by the Office upon request and payment of the
necessary fee.
[083] Fig. 1 depicts a schematic of antigen binding protein formats used in
Example 3
of the disclosure.
[084] Fig. 2 depicts in vitro cell killing in osteosarcoma cells incubated
with monovalent
pMTIC-targeting T cell engagers in formats 1 and 2 (Fig. 2A), and 3 and 4
(Fig. 2B). Fig 2C
depicts in vitro cell killing in osteosarcoma cells incubated with bivalent
pMHC-targeting T cell
engagers in formats 5 and 6. Fig. 2D depicts a direct comparison of in vitro
cell killing of
osteosarcoma cells mediated by monovalent and bivalent pMEIC-targeting T cell
engagers in
formats 3 and 6, respectively.
[085] Fig. 3 depicts percent cancer cell killing in osteosarcoma (Fig. 3A)
cells or
melanoma cells (Fig. 3B) incubated with a dual pMHC-targeting T cell engager
(squares)
compared to a single pMEIC-targeting T cell engager (circles). MAGE-A4 & HLA-
A*02:01
positive cell line U2OS (osteosarcoma) was incubated with human PBMCs at an
E:T ratio of 10:1.
Cancer cell killing was measured at various concentrations of the two antigen
binding proteins
with an LDH release assay after 48 hours. T cell activation was determined by
quantification of
CD69 and CD25 markers on the CD8 T cell population after 24h using flow
cytometry (C:
osteosarcoma cells; D: melanoma cells).
[086] Fig. 4 depicts a schematic of one embodiment of the bispecific antibody
of the
invention. The represented embodiment, a Fab-(scFv)2, comprises an anti-CD3
Fab fragment and
two single chain antibody fragments (scFv) which specifically bind target
peptides presented on
MEW complexes. The pMHC binding scFvs may be linked to the C-termini of the
CH1- and CL-
domains via a glycine-serine flexible linker.
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[087] Fig. 5 depicts percent cancer cell killing in osteosarcoma cells
incubated with a
dual pMHC-targeting T cell engager (circles) compared to a single pMFIC-
targeting T cell
engager (triangles) in Fab-(scFv)2 and Fab-scFv formats, respectively.
[088] Fig. 6 depicts percent cell survival in lung squamous cell carcinoma
(Fig. 6A) and
colorectal adenocarcinoma (Fig. 6B) cells incubated with two distinct pM_HC-
targeting T cell
engagers in mono- and dual formats.
[089] Fig. 7A and Fig. 7B depict graphs of in vitro cell killing with antigen
binding
proteins with MAGE-A4 binding arms comprising two identical VHHs (Fig. 7A) or
scFvs (Fig.
7B) fused to CD3 binding Fabs with low (circle), mid (square) and high
(triangle) affinities.
[090] Fig. 8 depicts cytokine release in antigen-positive osteosarcoma cells
incubated
with three different dual pMHC-targeting T cell engagers in Fab-VHEI2 format.
Each engager
has a different level of binding affinity for CD3 (high, mid, and low). MAGE-
A4 & TIL,A-A*02
positive cell lines were incubated with human PBMCs at an E:T ratio of 10:1.
Cytokines IL-2
(Fig. 8A) and IFN gamma (Fig. 8B) were measured at various concentrations of
the three antigen
binding proteins.
[091] Fig. 9 depicts in vitro cell killing of the dual pM_HC T cell engager
with low (41
nM) and high (0.1 nM) affinity to the cancer antigen MAGE-A4 and equal
affinity to CD3.
[092] Fig. 10 depicts a schematic of the exemplary bispecific antibody of the
invention
that binds to a T cell and two pMTIC targets on a tumor cell (Anti-MAGE-A4
Dual engager) and
a schematic of a comparator consisting of an affinity enhanced recombinant
soluble T-cell
receptor (sTCR) fused to an anti-CD3 fragment. The MAGE-A4 affinity indicated
for the Fab-
(scFv)2 was measured in monovalent format.
[093] Fig. 11A depicts in vitro T cell activation in TAP-deficient T2 cells
loaded with
HLA-A*02:01-restricted cancer target peptide MAGE-A4 and similar
physiologically relevant
off-target Si (GLADGRTHTV, SEQ ID NO.: 89) and S16 (GLYDGPVHEV, SEQ ID NO.:
90)
peptides upon co-culture with dual pMHC-targeting T-cell engager or a sTCRxCD3
comparator
and healthy donor PBMCs. Fig. 11B depicts IFN gamma release associated with T
cell activation
in T2 cells loaded with cancer target peptide MAGE-A4 and similar
physiologically relevant off-
target S I and SI6 peptides upon co-culture with dual pMHC-targeting T-cell
engager and healthy
donor PBMCs.
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[094] Fig. 12 shows that dual pMEIC-targeting T-cell engager demonstrates
limited
cross-reactivity towards antigen-negative cells in vitro. Percent cytotoxicity
was determined for
melanoma SK-MEL-30 cells (Fig. 12A), lung adenocarcinoma NCI-H441 cells (Fig.
12B), breast
cancer MDA-MB-231 cells (Fig. 12C) and pancreatic carcinoma PANC-1 cells (Fig.
12D).
[095] Fig. 13 depicts percent cancer cell killing in osteosarcoma cells and
melanoma
cells incubated with different concentrations of a dual pMHC-targeting T cell
engager or an
affinity enhanced recombinant sTCR T cell engager comparator shown in Fig. 10.
MAGE-A4 &
HLA-A*02:01 positive cell lines A375 (melanoma) and U2OS (osteosarcoma) were
incubated
with human PBMCs at an E:T ratio of 10:1. LDH release was measured as a marker
of cancer cell
killing at various concentrations of the two antigen binding proteins.
[096] Fig. 14 depicts cytokine release in osteosarcoma cells or melanoma cells
co-
cultured with PBMCs from healthy donors incubated with a dual pMEC-targeting T
cell engager
compared or the sTCR T cell engager comparator shown in Fig. 10. MAGE-A4 & HLA-
A*02
positive cell lines A375 (melanoma) and U2OS (osteosarcoma) were incubated
with human
PBMCs at an E:T ratio of 10:1. Cytokines IL-2 and IFN gamma were quantified
using ELISAs to
measure the level of cytokines released in the supernatant at various
concentrations of the two
antigen binding proteins.
[097] Fig. 15 depicts live cell imagining of MAGE-A4 positive NCI-H1703 lung
squamous carcinoma cells co-cultured with human PBMCs in presence of a dual
pM_HC-targeting
T-cell engager ("dual p1V1TIC TCE") with specificity for MAGE-A4/HLA-A*02:01.
Fig. 15A
(left) shows lung cancer cells and PBMCs alone; Fig. 15B (right) shows lung
cancer cells and
PBMCs in presence of the dual pl\TFIC TCE.
[098] Fig. 16 depicts detection of pre-existing anti-drug antibodies (ADAs)
against the
comparator and an antibody devoid of pre-existing ADA epitopes. The comparator
and the
antibody devoid of pre-existing ADA epitopes were evaluated in serum samples
from 10 healthy
naive Caucasian human donors. Pre-existing ADAs were detected by ELISA.
[099] Fig. 17 depicts detection of pre-existing ADAs in humanized single
domain
antibodies (sdAb) with select modifications. "+A" corresponds to the addition
of an alanine. "-S"
corresponds to the deletion of a serine at position 113, according to Kabat
numbering. "-SS"
corresponds to the deletion of a serine at position 112 and 113, according to
Kabat numbering.
"SSS" corresponds to the substitution of hydrophobic amino acids at Kabat
positions 11, 89, and
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108 to serine amino acids. The ADA response was measured with an ELISA over
different sample
serum concentrations.
[0100] Fig. 18 depicts detection of pre-existing ADAs in Fab scFy antigen
binding
proteins with selected modifications on the scFv binding arm. "+A" corresponds
to the addition
of an alanine. "-S" corresponds to the deletion of a serine at position 113,
according to Kabat
numbering. "-SS" corresponds to the deletion of a serine at position 112 and
113, according to
Kabat numbering. "SS S- corresponds to the substitution of hydrophobic amino
acids at Kabat
positions 11, 89, and 108 to serine amino acids. The ADA response was measured
with an ELISA
over different sample serum concentrations.
DETAILED DESCRIPTION
[0101] Generally, nomenclature used in connection with cell and tissue
culture, molecular
biology, immunology, microbiology, genetics and protein and nucleic acid
chemistry and
hybridization described herein is well-known and commonly used in the art. The
methods and
techniques provided herein are generally performed according to conventional
methods well
known in the art and as described in various general and more specific
references that are cited
and discussed throughout the present specification unless otherwise indicated.
Enzymatic
reactions and purification techniques are performed according to
manufacturer's specifications,
as commonly accomplished in the art or as described herein. The nomenclature
used in connection
with, and the laboratory procedures and techniques of, analytical chemistry,
synthetic organic
chemistry, and medicinal and pharmaceutical chemistry described herein is well-
known and
commonly used in the art. Standard techniques are used for chemical syntheses,
chemical
analyses, pharmaceutical preparation, formulation, and delivery, and treatment
of patients.
[0102] Unless otherwise defined herein, scientific and technical terms used
herein have
the meanings that are commonly understood by those of ordinary skill in the
art. In the event of
any latent ambiguity, definitions provided herein take precedent over any
dictionary or extrinsic
definition. Unless otherwise required by context, singular terms shall include
pluralities and plural
terms shall include the singular. The use of the term "including," as well as
other forms, such as
"includes" and "included," is not limiting.
[0103] So that the invention may be more readily understood, certain terms are
first
defined.
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Antigen Binding Proteins
[0104] As used herein, the term "antibody" or "antigen binding protein" refers
to an
immunoglobulin molecule or immunoglobulin derived molecule that specifically
binds to, or is
immunologically reactive with an antigen or epitope, and includes both
polyclonal and
monoclonal antibodies, as well as functional antibody fragments, including but
not limited to
fragment antigen-binding (Fab) fragments, F(abl)2 fragments, Fab fragments,
FAT fragments,
recombinant IgG (rIgG) fragments, single chain variable fragments (scFv) and
single domain
antibodies (e.g., sdAb, sdFv, nanobody, VI-111) fragments. The antibody may
thus be a single
domain antibody or comprise at least one variable light and at least one
variable heavy chain. In
one embodiment, the at least one variable light and at least one variable
heavy chain are displayed
as a single polypeptide chain. The term "antibody" or "antigen binding
protein" includes germline
derived antibodies. The term "antibody' or -antigen binding protein" includes
genetically
engineered or otherwise modified forms of immunoglobulins, such as
intrabodies, peptibodies,
chimeric antibodies, fully human antibodies, humanized antibodies,
heteroconjugate antibodies
(e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-
scFv, tandem tri-scFv)
and the like. Unless otherwise stated, the term "antibody" or "antigen binding
protein" should be
understood to encompass functional antibody fragments thereof.
[0105] In certain embodiments, the antigen binding protein is not a T cell
receptor (TCR),
including but not limited to, a soluble TCR.
[0106] In certain embodiments, the antigen binding protein is multispecific
(i.e., binds to
two or more different target molecules or to two or more epitopes on the same
target molecule).
In certain embodiments, the antigen binding protein is bispecific and e.g.,
binds to two different
target molecules or to two epitopes on the same target molecule. In certain
embodiments, the
antibody is trispecific and e.g., binds to at least three different target
molecules.
[0107] The antigen binding protein may be monovalent or multivalent, i.e.,
having one or
more antigen binding sites. Non-limiting examples of monovalent antigen
binding proteins
include scFv, Fab, scFab, dAb, V.HIH, V(NAR), DARPins, affilins and
nanobodies. A multivalent
antigen binding protein can have two, three, four or more antigen binding
sites. Non-limiting
examples of multivalent antigen binding proteins include full-length
immunoglobulins,
F(ab1)2fragments, bis-scFv (or tandem scFv or BiTE), DART, diabodies, scDb,
DVD-Ig, IgG-
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scFab, scFab-Fc-scFab, IgG-scFv, scFv-Fc, scFv-fc-scFv, Fv2-Fc, FynomABs,
quadroma,
CrossMab, DuoBody, triabodies and tetrabodies. In some embodiments, the
multivalent antigen
binding protein is bivalent, i.e., two binding sites are present. In some
embodiments, the
multivalent antigen binding protein is bispecific, i.e., the antigen binding
protein is directed
against two different targets or two different target sites on one target
molecule. In some
embodiments, the multivalent antigen binding protein includes more than two,
e.g., three or four
different binding sites for three or four, respectively, different antigens.
Such antigen binding
protein is multivalent and multispecific, in particular tri- or tetra-
specific, respectively.
[0108] In some embodiments, the antigen binding proteins are multispecific
(e.g.,
bispecific), such as, without being limited to, diabodies, single-chain
diabodies, DARTs, BiTEs,
tandem scFvs or IgG-like asymmetric heterobispecific antibodies. In certain
embodiments, one or
the binding specificities of the multispecific antigen binding protein is an
immune cell engager
(i.e., comprising binding affinity to a cell surface protein of an immune
cell). Examples of immune
cells that may he recruited include, hut are not limited to, T cells, B cells,
natural killer (NT() cells,
natural killer T (NKT) cells, neutrophil cells, monocytes, and macrophages.
Examples of surface
proteins that may be used to recruit immune cells includes, but are limited
to, CD3, TCRot, TCRP,
CD16, NKG2D, CD89, CD64, and CD32.
[0109] In certain embodiments, the immune cell target antigen is CD3. The term
CD3
refers to the cluster of differentiation 3 co-receptor (or co-receptor
complex) of the T cell receptor.
[0110] As used herein, a "single-chain variable fragment" (scFv) is an antigen
binding
protein comprising a heavy chain variable domain (VH) linked to a light chain
variable domain
(VL). The VH and VL domains of the scFv are linked via any appropriate art
recognized linker.
Such linkers include, but are not limited to, repeated GGGGS (SEQ ID NO.: 88)
amino acid
sequences or variants thereof. The scFv is generally free of antibody constant
domain regions,
although an scFv of the disclosure may be linked or attached to antibody
constant domain regions
(e g , antibody Fc domain) to alter various properties of the scFv, including,
but not limited to,
increased serum or tissue half-life. An scFv generally has a molecular weight
of about 25 kDa and
a hydrodynamic radius of about 2.5 nm.
[0111] As used herein, a "Fab fragment" or "Fab" or "Fab domain" is an
antibody
fragment comprising a light chain fragment comprising a variable light (VL)
domain and a
constant domain of the light chain (CL), and variable heavy (VH) domain and a
first constant
domain (CH1) of the heavy chain.
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[0112] As used herein, a "VHH", "nanobody", "heavy-chain only antibody",
"single
domain antibody", or "sdAb" is an antigen binding protein comprising a single
heavy chain
variable domain derived from the species of the Camelidae family, which
includes camels, llama,
alpaca. A VHH generally has a molecular weight of about 15 kDa.
[0113] The antigen binding proteins of the disclosure may comprise one or more
linkers
for linking the domains of the antigen binding protein (e.g., linking a VH and
VL to form a scFv,
or linking multiple binding domains to form a multispecific antigen binding
protein).
[0114] Illustrative examples of linkers include glycine polymers (Gly).;
glYcine-serine
polymers (GlynSer)., where n is an integer of at least one, two, three, four,
five, six, seven, or
eight; glycine-alanine polymers; alanine-serine polymers; and other flexible
linkers known in the
art.
[0115] Glycine and glycine-serine polymers are relatively unstructured, and
therefore may
be able to serve as a neutral tether between domains of fusion proteins such
as the antigen binding
proteins described herein. Glycine accesses significantly more phi-psi space
than other small side
chain amino acids, and is much less restricted than residues with longer side
chains (Scheraga,
Rev. Computational Chem. 1: 1173-142 (1992)). A person skilled in the art will
recognize that
design of an antigen binding protein in particular embodiments can include
linkers that are all or
partially flexible, such that the linker can include flexible linker stretches
as well as one or more
stretches that confer less flexibility to provide a desired structure.
[0116] Linker sequences can however be chosen to resemble natural linker
sequences, for
example, using the amino acid stretches corresponding to the beginning of
human CH1 and CI<
sequences or amino acid stretches corresponding to a portion of the hinge
region of human IgG.
[0117] The design of the peptide linkers connecting VL and VH domains in the
scFv
moieties are flexible linkers generally composed of small, non-polar or polar
residues such as,
e.g., Gly, Ser and Thr. A particularly exemplary linker connecting the
variable domains of the
scFv moieties is the (Gly4Ser)4 linker, where 4 is the exemplary number of
repeats of the motif.
[0118] Linkers connecting the scFv antigen binding proteins to the Fab domain
are also
envisioned. In certain embodiments, the scFv antigen binding proteins are
linked to the CH1 and
CL domains of the Fab with a Gly-Ser linker. In certain embodiments, the
linker comprises the
amino acid sequence GGGGS (SEQ ID NO.: 88).
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[0119] Other exemplary linkers include, but are not limited to the following
amino acid
sequences: GGG; DGGGS (SEQ ID NO.: 91); TGEKP (SEQ ID NO.. 92) (Liu et al,
Proc. Natl.
Acad. Sci.94: 5525-5530 (1997)); GGRR (SEQ ID NO.: 93); (GGGGS)i, (SEQ ID NO.:
88)
wherein n = 1, 2, 3, 4 or 5 (Kim et al, Proc. Natl. Acad. Sci.93: 1156-1160
(1996));
EGKSSGSGSESKVD (SEQ ID NO.: 94) (Chaudhary et al., Proc. Natl. Acad. Sci. 87:
1066-1070
(1990)); KESCISVSSEQLAQFRSLD (SEQ ID NO.: 95) (Bird et al., Science 242:423-
426
(1988)), GGRRGGGS (SEQ ID NO.: 96); LRQRDGERP (SEQ ID NO.: 97); LRQKDGGGSERP
(SEQ ID NO.: 98); and GSTSGSGKPGSGEGSTKG (SEQ ID NO.: 99) (Cooper et al,
Blood,
101(4): 1637-1644 (2003)). Alternatively, flexible linkers can be rationally
designed using a
computer program capable of modeling the 3D structure of proteins and peptides
or by phage
display methods.
[0120] The antibodies may comprise a variable light (VL) domain and a variable
heavy
(VH) domain. Each VL and VH domain further comprises a set of three CDRs.
[0121] As used herein, the term "complementarity determining region- or "CDR-
refers
to non-contiguous sequences of amino acids within antibody variable regions,
which confer
antigen specificity and binding affinity. In general, there are three CDRs in
each heavy chain
variable domain (CDRH1, CDRH2, CDRH3) and three CDRs in each light chain
variable domain
(CDRL1, CDRL2, CDRL3). "Framework regions" or "FRs" are known in the art to
refer to the
non-CDR portions of the variable domains of the heavy and light chains. In
general, there are four
FRs in each heavy chain variable domain (HFR1, HFR2, HFR3, and FIFR4), and
four FRs in each
light chain variable domain (LFR1, LFR2, LFR3, and LFR4). Accordingly, an
antibody variable
region amino acid sequence can be represented by the formula FR1-CDR1-FR2-CDR2-
FR3-
CDR3-FR4. Each segment of the formula, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3,
and FR4,
represents a discrete amino acid sequence (or a polynucleotide sequence
encoding the same) that
can be mutated, including one or more amino acid substitutions, deletions, and
insertions. In
certain embodiments, an antibody variable light chain amino acid sequence can
be represented by
the formula LFR1-CDRL I -LFR2-CDRL2-LFR3-CDRL3-LFR4. In certain embodiments,
an
antibody variable heavy chain amino acid sequence can be represented by the
formula FIFRI-
CDRH1-11FR2-CDRH2-1-1F'R3-CDRH3-HFR4.
[0122] The precise amino acid sequence boundaries of a given CDR or FR can be
readily
determined using any of a number of well-known schemes, including those
described by Kabat et
al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public
Health Service,
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National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme), Al-
Lazikani et al.,
(1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J.
Mol. Biol.
262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and
binding site
topography," J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme), Lefranc
M P et al.,
"IMGT unique numbering for immunoglobulin and T cell receptor variable domains
and Ig
superfamily V-like domains," Dev Comp Immunol, 2003 January; 27(1):55-77 (-
IMGT"
numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering
scheme for
immunoglobulin variable domains: an automatic modeling and analysis tool," J
Mol Biol, 2001
Jun. 8; 309(3):657-70, ("Aho" numbering scheme).
[0123] The boundaries of a given CDR or FR may vary depending on the scheme
used for
identification. For example, the Kabat scheme is based on sequence alignments,
while the Chothia
scheme is based on structural information. Numbering for both the Kabat and
Chothia schemes is
based upon the most common antibody region sequence lengths, with insertions
accommodated
by insertion letters, for example, "30a," and deletions appearing in some
antibodies The two
schemes place certain insertions and deletions ("indels") at different
positions, resulting in
differential numbering. The Contact scheme is based on analysis of complex
crystal structures
and is similar in many respects to the Chothia numbering scheme.
[0124] Table 1, below, lists exemplary position boundaries of CDRL1, CDRL2,
CDRL3
and CDRH1, CDRH2, CDRH3 of an antibody, as identified by Kabat, Chothia, and
Contact
schemes, respectively. For CDRH1, residue numbering is listed using both the
Kabat and Chothia
numbering schemes. CDRs are located between FRs, for example, with CDRL1
located between
LFR1 and LFR2, and so forth. It is noted that because the shown Kabat
numbering scheme places
insertions at H35A and H35B, the end of the Chothia CDRH1 loop when numbered
using the
shown Kabat numbering convention varies between H32 and H34, depending on the
length of the
loop.
Table 1 - Exemplary Position Boundaries of CDRs
CDR Kabat Chothia
Contact
LCDR1 L24-L34 L24-L34 L30-
L36
LCDR2 L50-L56 L50-L56 L46-
L55
LCDR3 L89-L97 L89-L97 L89-
L96
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HCDR1
H31¨H35B H26¨H32..34 H30¨H35B
(Kabat Numbering')
HCDR1
H31¨H35 H26¨H32 H30¨H35
(Chothia Numb ering2)
HCDR2 I-150¨H65 H52-1156 H47-1158
HCDR3 H95¨H102 H95¨H102 H93¨H101
1 ¨ Kabat et al. (1991), "Sequences of Proteins of Immunological Interest,"
5th Ed. Public Health Service,
National Institutes of Health, Bethesda, MD
2 ¨ Al-Lazikani et al. (1997), J. Alol. Biol. 273:927-948
[0125] Thus, unless otherwise specified, a "CDR" or "complementary determining
region," or individual specified CDRs (e.g., CDRH1, CDRH2), of a given
antibody or fragment
thereof, such as a variable domain thereof, should be understood to encompass
a (or the specific)
complementary determining region as defined by any of the known schemes.
Likewise, unless
otherwise specified, an "FR" or "framework region," or individual specified
FRs (e.g., "HFR1,"
"HFR2") of a given antibody or fragment thereof, such as a variable domain
thereof, should be
understood to encompass a (or the specific) framework region as defined by any
of the known
schemes. In some instances, the scheme for identification of a particular CDR
or FR is specified,
such as the CDR as defined by the Kabat, Chothia, or Contact method. In other
cases, the
particular amino acid sequence of a CDR or FR is given.
[0126] In certain embodiments, the antigen binding proteins disclosed here are
rabbit
antigen binding proteins or rabbit-derived antigen binding proteins. In
certain embodiments, the
rabbit antigen binding proteins are humanized. As used herein, the term
"humanized" or
"humanization" refers to an antigen binding protein that has been altered to
make it more like a
human antibody. Non-human antigen binding proteins, such as rabbit antigen
binding proteins,
would elicit a negative immune reaction if administered to a human for
therapy. It is therefore
advantageous to humanize the rabbit antigen binding proteins for later
therapeutic use.
[0127] In certain embodiments, the antigen binding proteins are humanized
through
resurfacing (i.e., remodel the solvent-accessible residues of the non-human
framework such that
they become more human-like). Resurfacing strategies are described in more
detail in
W02004/016740, W02008/144757, and W02005/016950, each of which is incorporated
herein
by reference.
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[0128] In certain embodiments, the antigen binding proteins are humanized
through CDR
grafting (i.e., inserting the rabbit antigen binding protein CDRs into a human
antibody acceptor
framework). Grafting strategies and human acceptor frameworks are described in
more detail in
W02009/155726, incorporated herein by reference.
[0129] As used herein, the term "affinity" (or "binding affinity" as used
interchangeably
herein) refers to the strength of the interaction between an antibody's
antigen binding site and the
epitope to which it binds. As readily understood by those skilled in the art,
an antibody or antigen
binding protein affinity may be reported as an equilibrium dissociation
constant (KD) in molarity
(M). The equilibrium dissociation constant KDis calculated from the
association rate constant ka
(having the unit M's') and the dissociation rate constant ka (having the unit
s-1) by ka/ka. The
antibodies of the disclosure may have KD values in the range of 10-7 to 10' M.
High affinity
antibodies have KD values of 10-9 M (1 nanomolar, nM) and lower. For example,
a high affinity
antibody may have a KD value in the range of about 1 nM to about 0.01 nM. A
high affinity
antibody may have Ku value of about 1 nM, about 0.9 nM, about 0.8 nM, about
0.7 nM, about 0.6
nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, or about 0.1 n1\4.
Very high affinity
antibodies have KD values of 1012 M (1 picomolar, pM) and lower. Weak, or low,
affinity
antibodies may have KD values in the range of 10-1 to 10-4 M. Low affinity
antibodies may have
KD values of 10-4 M and higher, such as 10-4 M, 10-3 M, 10-2 M, or 10-1 M. The
ability of an
antibody to bind to a specific antigenic determinant (e.g., a target peptide-
MHC) can be measured
either through an enzyme-linked immunosorbent assay (ELISA) or other
techniques familiar to
one of skill in the art, e.g., surface plasmon resonance (SPR) technique
(analyzed on a BIAcore
instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional
binding assays (Heeley,
Endocr Res 28, 217-229 (2002)). Generally, binding parameters can be
determined at
temperatures in the range of 20 C to 30 C. The present specification makes
reference to binding
parameters measured by SPR throughout. Typically, in embodiments pertaining to
each reference
to SPR throughout the present specification, association rate constant values,
dissociation rate
constant values and equilibrium dissociation constant values recited herein
are determined by SPR
at 25 C. Preferably, the SPR-based system used is the BIAcore SPR system. The
skilled person
will appreciate that the binding parameters can be measured in the context of
the monovalent or
bivalent bi-, tri- or multispecific constructs; preferably, the parameters are
determined in the
context of the whole construct.
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[0130] As used herein, the term "T cell receptor" or "TCR" refers to a
heterodimeric
protein comprised of two different chains (TCRa and TCR), which structurally
belong to the
immunoglobulin (Ig) superfamily. The extracellular portion of each chain is
composed of variable
("Va" and "VP") and constant ("Ca" and "Co") domains, and a hinge region,
where the formation
of a stabilizing disulfide bond occurs. The intracellular region forms a non-
covalent interaction
with another trans-membrane protein, CD3, which in the case of the correct
target recognition
leads to a series of conformational changes and a first T cell activation
signal. Recognition and
binding of peptide-MIIC (Pmhc) by a TCR is governed by the six hypervariable
loops, termed
complementarity determining regions (CDRs), located on the variable domains of
the TCRa
(CDRal, CDRa2, CDRa3) and TCR P (CDRP1, CDR(32, CDR133). CDR3 loops (CDRa3 and
CDRP3) lead the recognition of the processed antigen with the support of CDRal
and CDRP1,
that have been implicated in the recognition of the N- and C-terminal amino
acids of the presented
peptide, respectively (Rudolph et al Annu Rev Immunol. 24:419-66. 2006).
Recognition of the
MEW is typically achieved through the interaction with CDRa2 and CDRP2. The
high sequence
diversity of the TCR is achieved through V(D)J recombination process, in which
the variable
domain is generated from a combination of genes: V (variable) and J (joining)
for both TCRa and
TCRP, and an additional D (diversity) gene for TCRP. The high antigen
specificity of the TCR
is controlled by the thymic maturation process, in which the self-reacting T
cells are negatively
selected TCR affinity towards the specific pMHC and the functional avidity are
the key factors
controlling T-cell activation. A critical role in antigen recognition,
however, is played by the
affinity (KD), i.e., the strength of binding between the TCR and the cell-
displayed pMHC (Tian
et al. J Immunol. 179:2952-2960. 2007). The physiological affinities of TCRs
range from 1 mM
to 100 m1\4 (Davis et al. Annu Rev Immunol. 16:523-544. 1998), which, in
comparison to
antibodies, is relatively low.
[0131] As used herein, the term "peptide-MT-IC" refers to a major hi
stocompatibility
complex (MI-1C) molecule (MHC-I or -II) with an antigenic peptide bound in a
peptide binding
pocket of the MHC. In certain embodiments, the MHC is a human MHC.
Dual Peptide-MHC ¨ Immune Cell Engaging Antigen Binding Proteins
[0132] Certain antigen binding proteins described herein possess at least two
pMTIC
binding domains and a binding domain with binding specificity to a cell
surface protein of an
immune cell (e.g., CD3 on the surface of a T cell; an "immune cell binding
domain"). Targeting
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two pMHC complexes on the surface of a target cell (e.g., a cancer cell)
improves target cell
engagement through avidity-enhanced binding. The enhanced binding (i.e., lower
apparent KD of
the multivalent interaction) may in turn promote improved target cell killing
relative to an antigen
binding protein that only has one pM1HC binding domain. The avidity-enhanced
binding created
by at least two pMHC binding domains may be particularly useful when targeting
pMHC
complexes of low copy number on the surface of a target cell (e.g., cancer
cell).
[0133] In one aspect, the disclosure provides an antigen binding protein
comprising: a) a
Fab domain which specifically binds to a cell surface protein of an immune
cell, the Fab domain
comprising a heavy chain and a light chain; b) at least a first pMFICbinding
domain operably
linked to the heavy chain, wherein the first pMHC binding domain binds to
first target peptide-
MEW (pMT1C) complex; and c) at least a second pMHC binding domain operably
linked to the
light chain, wherein the second pMHC binding domain binds to a second pMHC
complex.
[0134] In certain embodiments, the Fab domain heavy chain comprises a CHI
domain and
a VH domain. In certain embodiments, the Fab domain further comprises at least
5 amino acids
of an antibody hinge region, in particular at the C-terminus of the heavy
chain of the Fab domain.
In certain embodiments, said Fab domain comprises up to, or at most, 10 amino
acids of an
antibody hinge region. In certain embodiments, the Fab domain comprises the
sequence stretch
up to the first cysteine of the antibody hinge region. In certain embodiments,
said sequence stretch
is or comprises the sequence EPKSC (SEQ ID NO.: 87). The presence of cysteine
allows for an
additional disulfide bridge which may further stabilize the antigen binding
protein. In some
embodiments, said at most 10 amino acids of an antibody hinge region comprises
EPKSCDKTHT
(SEQ ID NO.: 100). The antibody hinge region may additionally comprise the
sequence GGGGS
(SEQ ID NO.: 88) which may serve as a linker sequence to the pMHC binding
domain(s). Thus,
in some embodiments, a pMHC binding domain is linked to the C-terminal end of
the Fab CH1
domain via any of EPKSCGGGGS (SEQ ID NO.: 101), EPKSCDKTHT (SEQ ID NO.: 100),
EPKSCDKTHTGGGGS (SEQ ID NO.: 102), DKTHT (SEQ ID NO.: 103), DKTHTGGGGS
(SEQ ID NO.: 104) or GGGGSGGGGS (SEQ ID NO.: 105) linker.
[0135] In certain embodiments, the Fab domain light chain comprises a CL
domain and a
VL domain. The CL domain may be followed by a linker, such as GGGGS (SEQ ID
NO.: 88).
[0136] Suitable linker sequences between the immune cell binding domain and
the pMHC
binding domains include glycine polymers (Gly)n; glycine-serine polymers
(GlynSer)y, wherein
n and y are an integer of at least one, two, three, four, five, six, seven, or
eight; glycine-alanine
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polymers; alanine-serine polymers; and other flexible linkers known in the
art. In various
embodiments, the linker sequence connecting the immune cell binding domain and
the pMHC
binding domain(s) is the (Gly4Ser)1 (SEQ ID NO.: 88) linker sequence.
[0137] In some embodiments, the antigen binding protein of the present
disclosure
comprises at least 5 amino acids of an antibody hinge region (in certain
embodiments the sequence
stretch up to the first cysteine of an antibody hinge region), such as 5-10
amino acids or at most
amino acids located at the C-terminus of the heavy chain of the Fab domain,
and further
comprises a sequence that follows the said at least 5 amino acids of an
antibody hinge region and
that serves as a linker connecting a first or second pMHC domain as described
elsewhere herein.
Preferably, the at least 5 amino acids of an antibody hinge region comprise
the sequence EPKSC
(SEQ ID NO.: 87), or comprise the sequence EPKSCDKTHT (SEQ ID NO.: 100), and
the
sequences that serve as a linker connecting the first or second pMHC binding
domain comprise
the linker sequences as described above. In some embodiments, the at least 5
amino acids of an
antibody hinge region comprise the sequence EPK SC (SEQ ID NO 87), or comprise
the
sequence EPKSCDKTHT (SEQ ID NO.: 100), and the sequences that serve as a
linker connecting
the first or second pMHC binding domain comprise the sequence GGGGS (SEQ ID
NO.: 88).
[0138] Linker sequences connecting the variable domains of an scFv may include
glycine
polymers (Gly)n; glycine-serine polymers (GlynSer)y, where n and y are
integers of at least one,
two, three, four, five, six, seven, or eight; glycine-alanine polymers;
alanine-serine polymers; and
other flexible linkers known in the art. In certain embodiments, the linker
sequence is a glycine-
serine linker sequence (GlynSer)y, where n and y are an integers of at least
one, two, three, four,
five, six, seven, or eight. In certain embodiments thereof, the linker
sequence connecting the
variable domains of an scFv is the (Gly4Ser)4 (SEQ ID NO.: 106) linker
sequence.
[0139] In certain embodiments, the antigen binding protein does not comprise
an Fe
domain. In certain embodiments thereof, such antigen binding protein lacking
an Fe domain is a
Fab-sdAb, a Fab-(sdAb)2, a Fab-scFv or a Fab-(scFv)2, a F(ab')2fragment, a bis-
scFv (or tandem
scFv or BiTE), a DART, diabodies, a scDb, a triabody, , a tetrabody, or MATCH.
[0140] In certain embodiments, the first target pMHC complex and the second
target
plVIFIC complex are the same (i.e., each complex comprises the same peptide
bound to the 1VIHC
molecule). In certain embodiments, the first pMHC binding domain and the
second pMHC
binding domain are the same (i.e., the binding domains bind to the same
epitope).
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[0141] In certain embodiments, the first target pMHC complex and the second
target
pMHC complex are different (i.e., each complex comprises a different peptide
bound to the MTIC
molecule). In certain embodiments, the first pMHC binding domain and the
second pMHC
binding domain are different (i.e., the binding domains bind to different
epitopes).
[0142] In certain embodiments, the first pMHC binding domain is operably
linked to the
C-terminus of the Fab heavy chain or the N-terminus of the Fab heavy chain. In
certain
embodiments, the first pMHC binding domain is operably linked to the C-
terminus of the Fab
light chain or the N-terminus of the Fab light chain.
[0143] In certain embodiments, the second pMEC binding domain is operably
linked to
the C-terminus of the Fab heavy chain or the N-terminus of the Fab heavy
chain. In certain
embodiments, the second pM_HC binding domain is operably linked to the C-
terminus of the Fab
light chain or the N-terminus of the Fab light chain.
[0144] In certain embodiments, the pMHC binding domain is a scFv or an sdAb.
As
described elsewhere herein, the pMHC binding domain may also be any one of a
scFab, a diabody,
or a Fab. As described elsewhere herein and as exemplified in the Examples and
the drawings,
the pMHC binding domain is in particular a scFv or a sdAb (VHH), more
particularly each of the
at least first pMHC binding domain and/or each of the at least second pMHC
binding domain is
scFv or a sdAb (VHH). Further, in accordance with the experimental data, in
certain
embodiments, both the at least first pMHC binding domain and the at least
second pMHC binding
domain are each a scFv, or are each a sdAb, and both the at least first pMHC
binding domain and
the at least second pMHC binding domain are the same. Still further, and as
exemplified in the
Examples and the drawings, in certain embodiments thereof, the antigen binding
protein is
bivalent for the target pMFIC complex and comprises no more than two pMHC
binding domains
and both said pM_LIC binding domains are targeting the same pMHC complex.
[0145] As described elsewhere herein, and as follows from the above, the at
least first and
the at least second pMHC binding domain may both be linked to either the heavy
chain, or may
both be linked to the light chain of the Fab domain. As further described
elsewhere herein and as
exemplified in the Examples and the drawings, in some embodiments, the at
least first and the at
least second pMHC binding domain are not linked to the same chain of the Fab
domain, i.e., one
is linked to the heavy chain of the Fab domain, and the other is linked to the
light chain of the Fab
domain. As described elsewhere herein, in certain embodiments,
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(i) the at least first pMHC binding domain is operably linked to the C-
terminus of
the heavy chain of the Fab domain, and the at least second pMHC binding domain
is operably linked to the C-terminus of the light chain of the Fab domain, or
(ii) the at least first pMHC binding domain is operably linked to the C-
terminus of
the heavy chain of the Fab domain, and the at least second pMHC binding domain
is operably linked to the N-terminus of the light chain of the Fab domain, or
(iii) the at least first pMHC binding domain is operably linked to the N-
terminus
of the heavy chain of the Fab domain, and the at least second pMHC binding
domain is operably linked to the N-terminus of the light chain of the Fab
domain,
or
(iv) the at least first pMFIC binding domain is operably linked to the N-
terminus
of the heavy chain of the Fab domain, and the at least second plVIFIC binding
domain is operably linked to the C-terminus of the light chain of the Fab
domain,
or
(v) the at least first pMHC binding domain is operably linked to the N-
terminus of
the heavy chain of the Fab domain, and the at least second pMHC binding domain
is operably linked to the C-terminus of the heavy chain of the Fab domain, or
(vi) the at least first pMHC binding domain is operably linked to the N-
terminus
of the light chain of the Fab domain, and the at least second pMHC binding
domain
is operably linked to the C-terminus of the light chain of the Fab domain, or
(vii) both the at least first pMHC binding domain and the at least second
plVfHC
binding domain are operably linked to the C-terminus or to the N-terminus of
the
light chain of the Fab domain, or
(viii) both the at least first pMHC binding domain and the at least second
pMHC
binding domain are operably linked to the C-terminus or to the N-terminus of
the
heavy chain of the Fab domain.
[0146] In certain embodiments, the antigen binding protein comprises:
1) a first pMHC binding scFv linked to the C-terminus of the Fab domain heavy
chain and a second pMHC binding scFv linked to the C-terminus of the Fab
domain light chain,
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2) a first pMHC binding scFv linked to the N-terminus of the Fab
domain heavy chain and a second pMHC binding scFv linked to the N-
terminus of the Fab domain light chain;
3) a first pMHC binding scFv linked to the N-terminus of the Fab
domain heavy chain and a second pMHC binding scFv linked to the C-terminus
of the Fab domain light chain;
4) a first pMHC binding scFv linked to the C-terminus of the Fab
domain heavy chain and a second pMilIC binding scFv linked to the N-
terminus of the Fab domain light chain;
5) a first pMHC binding sdAb linked to the N-terminus of the Fab
domain heavy chain and a second pMHC binding sdAb linked to the N-
terminus of the Fab domain light chain;
6) a first pMHC binding sdAb linked to the C-terminus of the Fab
domain heavy chain and a second pMHC binding sdAb linked to the C-
terminus of the Fab domain light chain;
7) a first pM_HC binding sdAb linked to the N-terminus of the Fab
domain heavy chain and a second plVIFIC binding sdAb linked to the C-
terminus of the Fab domain light chain; or
8) a first pMHC binding sdAb linked to the C-terminus of the Fab
domain heavy chain and a second pMHC binding sdAb linked to the N-
terminus of the Fab domain light chain.
[0147] As described elsewhere herein and as exemplified in the Examples and
the
drawings, in certain embodiments, (i) the at least first pMHC binding domain
is operably linked
to the C-terminus of the heavy chain of the Fab domain, and the at least
second pMHC binding
domain is operably linked to the C-terminus of the light chain of the Fab
domain, or (ii) the at
least first pMHC binding domain is operably linked to the C-terminus of the
heavy chain of the
Fab domain, and the at least second pMHC binding domain is operably linked to
the N-terminus
of the light chain of the Fab domain. As further described elsewhere herein
and as further
exemplified in the Examples and the drawings, in certain embodiments thereof,
such antigen
binding protein has no more than two pMHC binding domains, i.e., is limited
with regard to
pMIFIC binding domains to one first plVIFIC binding domain and one second
plVfHC binding
domain. In certain embodiments thereof, the antigen binding protein is
bivalent for the target
pMHC complex. Accordingly, in such embodiments, the antigen binding protein
comprises no
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more than two pMHC binding domains, which are both binding to a pMHC complex,
which
comprises the same target peptide bound to/presented by the MEC molecule. As
further described
elsewhere herein and as further exemplified in the Examples and the drawings,
such antigen
binding protein is bispecific, and has preferably binding specificity for CD3.
In the
aforementioned embodiments, it is further preferred that the two pMHC binding
domains are both
scFv, or are both sdAb (VHI-1). Accordingly, the present disclosure
encompasses, in certain
embodiments, a bispecific bivalent antigen binding protein, comprising a Fab
domain which
specifically binds to CD3; and no more than two pMIIC binding domains, wherein
both plVIIIC
binding domains are targeting the same pMHC complex (i.e., the antigen binding
protein is
bivalent with regard to the target pMHC complex, wherein both pMHC binding
domains are each
a scFv, or are each a sdAb (VHH), and wherein (i) one of the two pMHC binding
domains is
operably linked to the C-terminus of the heavy chain of the CD3 binding
domain, and the other
pMHC binding domain is operably linked to the C-terminus of the light chain of
the CD3 binding
domain, or (ii) one of the two pMHC binding domains is operably linked to the
C-terminus of the
heavy chain of the CD3 binding domain, and the other pMHC binding domain is
operably linked
to the N-terminus of the light chain of the CD3 binding domain. As described
elsewhere herein,
the two plVIEIC binding domains may also be any one of a scFab, a diabody or a
Fab.
[0148] As further described elsewhere herein and as further exemplified in the
Examples
and the drawings, in some embodiments, the antigen binding protein has no more
than two pMHC
binding domains, i.e., is limited with regard to pMHC binding domains to one
first pMEC binding
domain one second pMHC binding domain, in particular to pMHC binding domains
which are
both scFv and are the same (or are both sdAb and the same), and wherein one is
operably linked
to the heavy chain of the Fab domain, and the other is operably linked to the
light chain of the Fab
domain, wherein it is even more preferred that (i) one of the two pMHC binding
domains is
operably linked to the C-terminus of the Fab domain heavy chain, and the other
pMHC binding
domain is operably linked to the C-terminus of the Fab domain light chain, or
(ii) one of the two
pMHC binding domains is operably linked to the C-terminus of the Fab domain
heavy chain, and
the other pM1-IC binding domain is operably linked to the N-terminus of the
Fab domain light
chain.
[0149] In certain embodiments, the first pMHC binding domain and/or the second
pMHC
binding domain comprise a variable heavy chain having a polar amino acid at
position 11, 89
and/or 108, according to Kabat numbering. The presence of polar amino acids at
the indicated
positions may reduce anti-drug antibodies.
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[0150] In certain embodiments, the immune cell binding domain, such as the Fab
domain
and/or the CD3 binding domain described elsewhere herein, comprises a variable
heavy chain
having a non- polar amino acid at position 11, 89 and/or 108, according to
Kabat numbering.
[0151] In certain embodiments, the variable heavy chain comprises: leucine (L)
or serine
(S) at amino acid position 11, according to Kabat numbering; valine (V),
serine (S), or threonine
(T) at amino acid position 89, according to Kabat numbering; and/or leucine
(L), serine (S), or
threonine (T) amino acid position 108, according to Kabat numbering.
[0152] In certain embodiments, when leucine (L) is present at amino acid
position 11,
then serine (S) or threonine (T) are present at amino acid position 89, and
serine (S) or threonine
(T) are present at amino acid position 108, according to Kabat numbering.
[0153] In certain embodiments, when valine (V) is present at amino acid
position 89, then
serine (S) is present at amino acid position 11, and serine (S) or threonine
(T) are present at amino
acid position 108, according to Kabat numbering.
[0154] In certain embodiments, when leucine (L) is present at amino acid
position 108,
then serine (S) or threonine (T) are present at amino acid position 11, and
serine (S) or threonine
(T) are present at amino acid position 89, according to Kabat numbering.
[0155] In certain embodiments, the polar amino acid is serine (S) and/or
threonine (T).
[0156] In certain embodiments, the variable heavy chain comprises serine (S)
at amino
acid position 11, serine (S) or threonine (T) at amino acid position 89, and
serine (S) or threonine
(T) at amino acid position 108, according to Kabat numbering.
[0157] In certain embodiments, the variable heavy chain comprises serine (S)
at amino
acid position 11, serine (S) at amino acid position 89, and serine (S) at
amino acid position 108,
according to Kabat numbering.
[0158] In certain embodiments, the immune cell binding domain, in particular
when not
comprising a CH domain, i.e., not being a Fab domain, but e.g. a scFv or a
sdAb, comprises a
variable heavy chain having a serine (S) at position 113 deleted, according to
Kabat numbering.
[0159] In certain embodiments, the first pMHC binding domain and/or the second
pMHC
binding domain comprise a variable heavy chain having a serine (S) at position
113 deleted,
according to Kabat numbering.
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[0160] In certain embodiments, the immune cell binding domain, in particular
when not
comprising a CH domain, i.e., not being a Fab domain, but e.g. a scFy or a
sdAb, comprises a
variable heavy chain having a serine (S) at position 112 deleted and a serine
(S) at position 113
deleted, according to Kabat numbering. In certain embodiments, the first pMHC
binding domain
and/or the second pMHC binding domain comprise a variable heavy chain having a
serine (S) at
position 112 deleted and a serine (S) at position 113 deleted, according to
Kabat numbering.
[0161] In certain embodiments, the antigen binding protein comprises an Si
13A, 5113G,
or Si 13T substitution, according to Kabat numbering.
[0162] In certain embodiments, the antigen binding protein comprises an Si
13A, 5113G,
or Si 13T substitution, and wherein S112 is deleted, according to Kabat
numbering.
[0163] In certain embodiments, the antigen binding protein comprises an S112A,
S112G,
or Si 12T substitution, according to Kabat numbering.
[0164] In certain embodiments, the antigen binding protein comprises an Si
12A, 5112G,
or S112T substitution, and wherein S113 is deleted, according to Kabat
numbering.
[0165] pMHC binding domains may e.g., be generated using the library approach
as
described in W02022190007A1, which is hereby incorporated by reference.
[0166] In certain embodiments, the target pMHC binding domain specifically
targets an
MHC restricted peptide derived from a tumor antigen or a viral antigen.
[0167] In accordance with the present disclosure, an antigen binding protein
as provided
by the present disclosure, in particular the at least first and/or the at
least second pMHC binding
domain, is highly selective and does not bind to a different pMHC complex,
such as a pMHC
complex presenting a different peptide.
[0168] In certain embodiments, the cell surface protein of an immune cell is
selected from
the group consisting of CD3, TCRc, TCRI3, CD i6, NKG2D, CD89, CD64, and CD32a.
In certain
embodiments, the cell surface protein of an immune cell is CD3 (cluster of
differentiation 3 co-
receptor (or co-receptor complex) of the T cell receptor). The CD3 protein
complex is composed
of four distinct chains. In mammals, the complex contains a CD3y (gamma)
chain/subunit, a CD3.3
(delta) chain/subunit, and two CD3E (epsilon) chains/subunits. Reference to
CD3 as the cell
surface protein of an immune cell is made herein throughout, and CD3 is the
particularly preferred
cell surface protein as exemplified, inter alia, by the Examples. Disclosed
herein is that in various
aspects and embodiments that are described throughout the specification and
that are pertaining
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to the immune cell binding domain, in particular the Fab domain, that
specifically binds to CD3
on the surface of an immune cell, in particular a T cell), the Fab domain may
specifically bind to
the CD3y (gamma) domain/subunit, the CD3,3 (delta) chain/subunit, and/or a
CD3s (epsilon)
chain/subunit of CD3. Preferably, the immune cell binding domain may
specifically bind to a
CD3s (epsilon) chain/subunit of CD3.
[0169] Suitable anti-CD3 binding domains are known in the art, particularly T-
cell
activating CD3-epsilon binding domains. The terms "CD3 binding domain- and
"anti-CD3
binding domain" are used interchangeably herein. In certain embodiments of the
present
disclosure, the anti-CD3 binding domain is any one of antibodies SP34, 0kt3 or
UCHT1, or a
variant sequence thereof having at least about 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99%
identity thereto, while retaining the same specificity as its parent. SP34,
0kt3 or UCHT1 are
murine antibodies; for therapeutic applications, humanized versions of SP34,
0kt3 or UCHT1,
i.e., huSP34, hu0kt3 or huUCHT1, are preferred. In certain embodiments, the
humanized variant
sequence of SP34, 0kt3 or UCHT1 is optimized for use in Fab format For
example, the
humanized huSP34, hu0kt3 or huUCHT1 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or
more
substitutions while retaining selective binding to CD3. Exemplary CD3 binding
domains are
disclosed in U5675 0325, W02008079713, US7635475, W02005040220, US7728114,
W09404679, US7381803, W02008119567, W02014110601, W02014145806,
W02015095392, W02016086189 and/or W02019195535A1, each of which is
incorporated
herein by reference. In certain embodiments, the immune cell is selected from
the group consisting
of a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT)
cell, a neutrophil cell, a
monocyte, and a macrophage. In certain embodiments, the immune cell is a T
cell.
[0170] In one embodiment, the anti-CD3 binding domain comprises the HCDR
sequences
of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ ID NOs.: 79, 81
and 82 or a
variant sequence thereof, having 1, 2 or 3 substitutions while retaining
specific antigen binding.
In one embodiment, the LCDR1 sequence comprises 1 substitution and is SEQ ID
NO.: 80.
[0171] In one embodiment, the anti-CD3 binding domain comprises the HCDR
sequences
of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ ID NOs.: 80, 81
and 82 or a
variant sequence thereof, having 1, 2 or 3 substitutions while retaining
specific antigen binding
In one embodiment, the LCDR1 sequence comprises 1 substitution and is SEQ ID
NO.: 79.
[0172] In one embodiment, the anti-CD3 binding domain comprises the VL
sequence of
SEQ ID NO.: 83 and the VH sequence of SEQ ID NO.: 84 or a variant sequence
thereof, being at
3i
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least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to said
amino acid
sequences while retaining specific antigen binding. In certain embodiments,
the variant sequence
comprises 1, 2, 3_4, 5, 6, 7, 8, 9, or 10 substitutions with regard to the
parental amino acid
sequence, such as e.g., 1 substitution in the VL sequence and 4 substitutions
in the VH sequence.
[0173] In one embodiment, the anti-CD3 binding domain comprises the VL
sequence of
SEQ ID NO.: 85 and the VH sequence of SEQ ID NO.: 86 or a variant sequence
thereof, being at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to said
amino acid
sequences while retaining specific antigen binding. In certain embodiments,
the variant sequence
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions with regard to the
parental amino acid
sequence, such as e.g., 1 substitution in the VL sequence and 4 substitutions
in the VH sequence.
[0174] Accordingly, the present disclosure encompasses, in
certain embodiments,
a bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the HCDR sequences of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ
ID NOs.:
79, 81 and 82 or variants thereof; and no more than two pMHC binding domains
targeting the
same pMHC complex, wherein both pMHC binding domains are each a scFv, such as
e.g., a Fab-
(scFv)2. Accordingly, the present disclosure encompasses, in certain
embodiments, a bispecific
bivalent antigen binding protein, comprising an anti-CD3-binding domain
comprising the HCDR
sequences of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ ID NOs.:
80, 81 and
82 or variants thereof; and no more than two pMHC binding domains targeting
the same pMHC
complex, wherein both plVLEIC binding domains are each a scFv, such as e.g., a
Fab-(scFv)2.
Accordingly, the present disclosure encompasses, in certain embodiments, a
bispecific bivalent
antigen binding protein, comprising an anti-CD3-binding domain comprising the
VL sequence of
SEQ ID NO.: 83 and the VH sequence of SEQ ID NO.: 84 or variants thereof; and
no more than
two pMHC binding domains targeting the same pMHC complex, wherein both pMHC
binding
domains are each a scFv, such as e.g., a Fab-(scFv)2. Accordingly, the present
disclosure
encompasses, in certain embodiments, a bispecific bivalent antigen binding
protein, comprising
an anti-CD3-binding domain comprising the VL sequence of SEQ ID NO.: 85 and
the VH
sequence of SEQ ID NO.: 86 or variants thereof; and no more than two pMHC
binding domains
targeting the same pMHC complex, wherein both pMHC binding domains are each a
scFv, such
as e.g., a Fab-(scFv)2. Accordingly, the present disclosure encompasses, in
certain embodiments,
a bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the HCDR sequences of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ
ID NOs.:
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79, 81 and 82 or variants thereof; and no more than two pMHC binding domains
targeting the
same pMHC complex, wherein both p1\4FIC binding domains are each a sdAb (VHH),
such as
e.g., a Fab-(sdAb)2. Accordingly, the present disclosure encompasses, in
certain embodiments, a
bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the HCDR sequences of SEQ ID NOs. : 76, 77 and 78 and the LCDR sequences of
SEQ ID NOs.:
80, 81 and 82 or variants thereof, and no more than two pMHC binding domains
targeting the
same pMHC complex, wherein both pMHC binding domains are each a sdAb (VHH),
such as
e.g., a Fab-(sdAb)2. Accordingly, the present disclosure encompasses, in
certain embodiments, a
bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the VL sequence of SEQ ID NO.: 83 and the VH sequence of SEQ ID NO.: 84 or
variants thereof,
and no more than two pMHC binding domains targeting the same p1\41-1C complex,
wherein both
pMHC binding domains are each a sdAb (VHH), such as e.g., a Fab-(sdAb)2.
Accordingly, the
present disclosure encompasses, in certain embodiments, a bispecific bivalent
antigen binding
protein, comprising an anti-CD3-binding domain comprising the VL sequence of
SEQ ID NO.:
85 and the VH sequence of SEQ ID NO.: 86 or variants thereof, and no more than
two pMHC
binding domains targeting the same plVITIC complex, wherein both pMHC binding
domains are
each a sdAb (VET), such as e.g., a Fab-(sdAb)2.
[0175] In certain embodiments, the immune cell binding domain, in particular
the Fab
domain, specifically binds to CD3 with a binding affinity (KD) between about 1
nM to about 150
nM (e.g., 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM,
12 nM, 13
nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24
nM, 25 n1\4,
26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM,
37 nM, 38
nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49
nM, 50 nM,
51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM,
62 nM, 63
nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74
nM, 75 nM,
76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 86 nM,
87 nM, 88
nM, 89 nM, 90 nM, 91 nM, 92 nM, 93 nM, 94 nM, 95 nM, 96 nM, 97 nM, 98 nM, 99
nM, 100
nM, 101 nM, 102 nM, 103 nM, 104 nM, 105 nM, 106 nM, 107 nM, 108 nM, 109 nM,
110 nM,
111 nM, 112 nM, 113 nM, 114 nM, 115 nM, 116 nM, 117 nM, 118 nM, 119 nM, 120
nM, 121
nM, 122 nM, 123 nM, 124 nM, 125 nM, 126 nM, 127 nM, 128 nM, 129 nM, 130 nM,
131 nM,
132 nM, 133 nM, 134 nM, 135 nM, 136 nM, 137 nM, 138 nM, 139 nM, 140 nM, 141
nM, 142
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nM, 143 nM, 144 nM, 145 nM, 146 nM, 147 nM, 148 nM, 149 nM, 150 nM), as
determined by
SPR. In certain embodiments, the immune cell binding domain, in particular the
Fab domain,
specifically binds to CD3 with a binding affinity (Kn) between about 1 nM to
about 50 nM, as
determined by SPR. In certain embodiments, the immune cell binding domain, in
particular the
Fab domain, specifically binds to CD3 with a binding affinity (KD) between
about 20 nM to about
50 nM, as determined by SPR.
[0176] In certain embodiments, the immune cell binding domain, in particular
the Fab
domain, specifically binds to CD3 with a binding affinity (KD) of about 1 nM,
of about 10 nM, or
of about 50 nM, as determined by SPR.
[0177] In some embodiments, the association rate constant ka of the anti-CD3
binding
domain is between about 1 x105 to about ix 107 M-ls-1, such as at least lx106
or at least
2x106 M's'.
[0178] In some embodiments, the dissociation rate constant kd of the anti-CD3
binding
domain is between about 1>< 10-1 to about 1 x106 s-1, such as at least 2><10-3
s-1, or at least 3 x10-3 s-
1 or at least 4x 10-3 s4. Without being bound to theory, a fast dissociation
rate, e.g., a kd-value of
2-3 x10-3 s-1, may lead to less T cell overactivation and in consequence, less
cytokine release.
[0179] In one embodiment, the association rate constant ka and/or the
dissociation rate
constant kd are equivalent or similar for both CD3-heterodimers CD3E7
(epsilon/gamma) and
CD3so (epsilon/delta), i.e., there is no significant difference for either the
ka or the kd or both of
the anti-CD3 binding domain to CD3c-y (epsilon/gamma) and CD3E6
(epsilon/delta) when
measured under the same conditions, in particular when determined by SPR at 25
C. In certain
embodiments thereof, the association rate constant ka and/or the dissociation
rate constant kd
values that are within 1 fold of each other, 1.5 fold of each other, 2-fold of
each other, 2.5-fold of
each other or 3-fold of each other, i.e., association rate constant ka values
of lx 105 M-1s-1 and
3,105m-1s-1.
[0180] In certain embodiments, the first pMHC binding domain and/or the second
pMHC
binding domain binds the target pMHC complex with a binding affinity (KO of
about 100 pM to
about 20 nM (e.g., about 100 pM, about 150 pM, about 200 pM, about 250 pM,
about 300 pM,
about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about
600 pM, about
650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM,
about 950
pM, about 1 nIVI (1,000 pM), about 2 nM, about 3 nM, about 4 nM, about 5 nM,
about 6 n1\4,
about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM,
about 13 nM,
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about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 n1\4, about 19
nM, or about 20
nM). In certain embodiments, the first pMHC binding domain and/or the second
pMHC binding
domain binds the target pMHC complex with a binding affinity (KD) of about 100
pM to about 1
nM. In certain embodiments, the first pMHC binding domain and/or the second
pMHC binding
domain binds the target pMHC complex with a binding affinity (KD) of about 100
pM to about
400 pM. In certain embodiments, the first pl\TFIC binding domain and/or the
second pMHC
binding domain binds the target pMHC complex with a binding affinity (KD) of
about 500 pM to
about 2 nM, or about 500 pM to about 3 nM, 500 pM to about 5 nM, or about 500
pM to about
n114, or about 100 pM to about 20 nM. In preferred embodiments, the said
binding affinities
are determined by SPR, as described elsewhere herein.
[0181] In some embodiments, the association rate constant ka of the p1\414C
binding
domain is between about 1 x 105 to about 1 x 107 M's', preferably between
about 0.5 x106 wris-1
to about 3 x106 such as at least 0.5 x106 at least lx106m-is-i,
at least 2x106
or at least 3x106 kris-1
[0182] In some embodiments, the dissociation rate constant kd of the p1\41-1C
binding
domain is between about 1 x10-1 to about lx 106 s1, such as between about lx
10-2 to about 1 x 10-
5S-1, such as at least 2x10-3 s-1, at least 4x10-3 s-1, at least 6x10-3 s-1,
at least 8x103 s-1, at least
2x104 s-1, at least 4x 10-4 s-1, at least 6x10-4 s-1 or at least 8x10-4
[0183] In certain embodiments, the antigen binding protein comprises a
molecular weight
of about 75 kDa to about 110 kDa (e.g., about 75 kDa, about 80 kDa, about 85
kDa, about 90
kDa, about 95 kDa, about 100 kDa, about 105 kDa or about 110 kDa). In certain
embodiments,
the antigen binding protein has increased serum half-life relative to an
antigen binding protein
with a molecular weight of less than about 60 kDa.
[0184] In certain embodiments, the antigen binding protein is a Fab-(scFv)2
and comprises
a single Fab domain which specifically binds to CD3, a first pMHC binding scFy
linked to the C-
terminus of the Fab domain heavy chain and a second pMHC binding scFy linked
to the C-
terminus of the Fab domain light chain, wherein both plVIFIC binding scFvs
bind to the same
target, such as a MAGE-A4 derived peptide presented on a HLA-A2 complex, and
the variable
heavy chain of both pIVIFIC binding scFvs optionally or additionally comprises
serine (S) at amino
acid position 11, serine (S) at amino acid position 89, and serine (S) at
amino acid position 108,
according to Kabat numbering. Advantageously, the Fab domain comprises the
first few amino
acids of the antibody hinge region up to the first cysteine.
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[0185] In certain embodiments, the antigen binding protein is a Fab-(scFv)2
and comprises
(i) a single Fab domain which specifically binds to CD3 with an affinity (Ku)
from about 1 nM to
about 50 nM, (ii) a first pMHC binding scFy linked to the C-terminus of the
Fab domain heavy
chain and (iii) a second pMHC binding scFy linked to the C-terminus of the Fab
domain light
chain, wherein both pMHC binding scFvs have a binding affinity (Ku) of about
500 pM to about
10nM to the target pM1-1C complex.
[0186] An advantage of the antigen binding protein scaffolds of the disclosure
is the
intermediate molecular size of approximately 75-110 kDa. Blinatumomab, a
bispecific T cell
engager (BiTE), has shown excellent results in patients with relapsed or
refractory acute
lymphoblastic leukemia. Because of its small size (60 kDa), blinatumomab is
characterized by a
short serum half-life of several hours, and therefore continuous infusion is
needed (see, U.S.
7,112,324 BO. The antigen binding proteins of the disclosure are expected to
have significantly
longer half-lives in comparison to smaller bispecific antibodies, such as
BiTEs like
blinatumomab, and thus, do not require continuous infusion due to their
favorable half-life An
intermediate sized molecule may avoid kidney clearance and provide a half-life
sufficient for
improved tumor accumulation. While the antigen binding proteins of the
disclosure have
increased plasma half-life compared to other small bispecific formats, they
still retain the tumor
penetration ability. On the other hand, the molecules of the instant
disclosure lacking an Fe
domain are expected to have a shorter half-life than larger molecules
including a Fe domain. A
prolonged half-life may overstimulate T cells and lead to T cell exhaustion.
Also, especially in
solid tumors, a large molecular weight may translate into a lower degree of
tumor penetration. In
some embodiments, the in vivo half-life is of about 7 days.
[0187] The Fab domain of the antigen binding protein of the disclosure may
serve as a
specific heterodimerization scaffold to which the additional pMTIC binding
domains are linked.
The natural and efficient heterodimerization properties of the heavy chain (Fd
fragment) and light
chain (L) of a Fab fragment makes the Fab fragment a useful scaffold.
Additional binding domains
may be in several different formats, including, but not limited to, another
Fab domain, a scFv, or
an sdAb
[0188] Each chain of the Fab fragment can be extended at the N- or C-terminus
with
additional binding domains. The chains may be co-expressed in mammalian cells,
where the host-
cell Binding immunoglobulin protein (BiP) chaperone drives the formation of
the heavy chain-
light chain heterodimer (Fd:L). These heterodimers are stable, with each of
the binders retaining
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their specific affinities. The two remaining pMHC binding domains may then be
fused as scFvs
or sdAbs to distinct Fab chains where each chain can be extended, e.g., at the
C-terminus with an
additional scFv or sdAb domain (see, for example, Schoonjans et al. J.
Immunology, 165(12):
7050-7057, 2000; Schoonjans et al. Biomolecular Engineering, 17: 193-202,
2001.) An additional
advantage of using Fabs as a heterodimerization unit is that Fab molecules are
abundantly present
in serum and therefore may be non-immunogenic when administered to a subject.
[0189] Based on, and in line with the overall disclosure content of the
present
specification, aspects and embodiments of the present invention include:
[0190] 1. An antigen binding protein comprising:
a) a Fab domain which specifically binds to a cell surface protein of an
immune
cell, the Fab domain comprising a heavy chain and a light chain; and
at least a first peptide-MHC (pMHC) binding domain and at least a second plVII-
1C
binding domain, wherein both the at least first and the at least second pMHC
binding domain are operably linked to the heavy chain of the Fab domain, and
wherein the first pMHC binding domain binds to a first target pIVILIC complex
and
the second pMHC binding domain binds to a second target pMHC complex;
wherein the at least first and/or the at least second pMHC binding domain is
each
any one of a scFv, a scFab, a diabody, a sdAb (VHH) or a Fab
or
b) a Fab domain which specifically binds to a cell surface protein of an
immune
cell, the Fab domain comprising a heavy chain and a light chain; and
at least a first peptide-MI-IC (pMHC) binding domain and at least a second
pMIHC
binding domain, wherein both the at least first and the at least second pMHC
binding domain are operably linked to the light chain of the Fab domain, and
wherein the first pMHC binding domain binds to a first target pMHC complex and
the second pMHC binding domain binds to a second target pMHC complex;
wherein the at least first and/or the at least second pMHC binding domain is
each
any one of a scFv, a scFab, a diabody, a sdAb (VHH) or a Fab.
[0191] 2. The antigen binding protein of embodiment [1], wherein the antigen
binding
domain comprises at least 5 amino acids of an antibody hinge region, located
at the C-terminus
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of the CH1 domain of the Fab domain which specifically binds to a cell surface
protein of an
immune cell.
[0192] 3. The antigen binding protein of the [1] or [2], wherein the at least
first target
pMEIC complex and the at least second target pMHC complex are the same or are
different.
Preferably, they are the same.
[0193] 4. The antigen binding protein of any one of the above [1] to [3],
wherein:
(i) the first pMHC binding domain is operably linked to the C-terminus of the
heavy chain, and the second pMHC binding domain is operably linked to the N-
terminus of the heavy chain; or
(ii) the first pMEIC binding domain is operably linked to the N-terminus of
the
heavy chain, and the second plVIFIC binding domain is operably linked to the C-
terminus of the heavy chain; or
(iii) the first pMTIC binding domain is operably linked to the C-terminus of
the
light chain, and the second pMTIC binding domain is operably linked to the N-
terminus of the light chain; or
(ii) the first pMEIC binding domain is operably linked to the N-terminus of
the
light chain, and the second pMEIC binding domain is operably linked to the C-
terminus of the light chain.
[0194] 5. In any one of the above [1] to [4], the Fab domain which
specifically binds to a
cell surface protein of an immune cell comprises a variable heavy chain having
a non-polar amino
acid at position 11, 89 and/or 108, according to Kabat numbering, as described
elsewhere herein.
[0195] 6. In any one of the above [1] to [5], the at least first and/or the at
least second
pMEIC binding domain a comprises a variable heavy chain having
(i) a polar amino acid as described elsewhere herein, such as serine, at
position 11,
89 and/or 108, according to Kabat numbering, as described elsewhere herein;
and/or
(ii) a deletion or substitution at position 112 and/or position 113, as
described
elsewhere herein.
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[0196] 7. The antigen binding protein of any one of the above [1] to [6],
wherein the at
least first and/or the at least second pMHC binding domain specifically
targets an MEC restricted
peptide derived of a tumor antigen or a viral antigen.
[0197] 8. The antigen binding protein of any one of the above [1] to [7],
wherein the cell
surface protein of an immune cell is CD3, and wherein the immune cell is a T
cell.
[0198] 9. The antigen binding protein of any one of the above [1] to [8],
wherein the Fab
domain specifically binds to CD3 with a binding affinity (KD) between about 1
nM to about 50
nM, as determined by SPR.
[0199] 10. The antigen binding protein of any one of the above [1] to [9],
wherein the at
least first pMHC binding domain and/or the at least second pMHC binding domain
binds the
target peptide pMHC complex with a binding affinity (KD) of about 100 pM to
about 20 nM. In
preferred embodiments, the at least first plVEFIC binding domain and/or the at
least second pMHC
binding domain binds the target peptide pMHC complex with a binding affinity
(KD) of about 500
pM to about 10 nIVI or of about 500 pM to about 5 nM.
[0200] 11. The antigen binding protein of any one of the above [1] to [10],
comprising a
molecular weight of about 75 kDa to about 110 kDa.
[0201] 12. The antigen binding protein of any one of the above [1] to [11],
wherein the
antigen binding protein has increased serum half-life relative to an antigen
binding protein with a
molecular weight of < about 60 kDa.
[0202] 13. A composition comprising the antigen binding protein of any one of
the above
[1] to [12], preferably the composition is a pharmaceutical composition.
[0203] 14. A method of treating cancer or a viral infection comprising the
step of
administering the antigen binding protein of any one of the above [1] to [12],
or the composition
of [13], to a patient in need thereof
[0204] 15. An antigen binding protein comprising:
a) a Fab domain which specifically binds CD3 on a T cell, the Fab domain
comprising a heavy chain and a light chain;
b) at least a first peptide-MHC (pMHC) binding domain operably linked to the C-
terminus of the heavy chain, wherein the first pMHC binding domain binds to a
first target peptide-MI-IC complex; and
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c) at least a second pMHC binding domain operably linked to the C-terminus of
the light chain, wherein the second pMHC binding domain binds to a second
target
pMHC complex,
wherein the at least first and/or the at least second pMHC binding domain is
each
any one of a scFv, a scFab, a diabody, a sdAb (VF1H), or a Fab.
[0205] 16. An antigen binding protein comprising:
a) a Fab domain which specifically binds CD3 on a T cell, the Fab domain
comprising a heavy chain and a light chain;
b) at least a first peptide-MI-IC (pMIIC) binding domain operably linked to
the C-
terminus of the heavy chain, wherein the first pMHC binding domain binds to a
first target peptide-MFIC complex; and
c) at least a second pMHC binding domain operably linked to the N-terminus of
the light chain, wherein the second pMHC binding domain binds to a second
target
pMHC complex,
wherein the at least first and/or the at least second pMHC binding domain is
each
any one of a scFv, a scFab, a diabody, a sdAb (VHH) or a Fab.
[0206] 17. The antigen binding protein of [15] or [16], wherein the antigen
binding
domain comprises at least 5 amino acids, optionally at most 10 amino acids, of
an antibody hinge
region located at the C-terminus of the CH1 domain of the Fab domain.
[0207] 18. The antigen binding protein of any one of the above [15] to [17],
wherein the
at least first target pMHC complex and the at least second target pMHC complex
are the same or
are different. Preferably, they are the same
[0208] 19. In any one of the above [15] to [18], the Fab domain comprises a
variable heavy
chain having a non-polar amino acid at position 11, 89 and/or 108, according
to Kabat numbering,
as described elsewhere herein.
[0209] 20. In any one of the above [15] to [19], the at least first and/or the
at least second
pMHC binding domain comprises a variable heavy chain having
(i) a polar amino acid as described elsewhere herein, e.g. serine, at position
11, 89
and/or 108, according to Kabat numbering, as described elsewhere herein;
and/or
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(ii) a deletion or substitution at position 112 and/or position 113, as
described
elsewhere herein.
[0210] 21. The antigen binding protein of any one of the above [15] to [20],
wherein the
at least first and/or the at least second pMHC binding domain specifically
targets an MEC
restricted peptide derived of a tumor antigen or a viral antigen.
[0211] 22. The antigen binding protein of any one of the above
[15] to [21], wherein
the Fab domain specifically binds to CD3 with a binding affinity (KD) between
about 1 nM to
about 50 nM, as determined by SPR.
[0212] 23. The antigen binding protein of any one of the above [15] to [22],
wherein the
at least first pMHC binding domain and/or the at least second pMHC binding
domain binds the
target peptide pMHC complex with a binding affinity (KD) of about 100 pM to
about 20 nM. In
preferred embodiments, the at least first plVEFIC binding domain and/or the at
least second pMHC
binding domain binds the target peptide pMHC complex with a binding affinity
(KD) of about 500
pM to about 10 nM or of about 500 pM to about 5 nM.
[0213] 24. The antigen binding protein of any one of the above [15] to [23],
comprising a
molecular weight of about 75 kDa to about 110 kDa.
[0214] 25. The antigen binding protein of any one of the above [15] to [24],
wherein the
antigen binding protein has increased serum half-life relative to an antigen
binding protein with a
molecular weight of less than about 60 kDa.
[0215] 26. A composition comprising the antigen binding protein of any one of
the above
[15] to [25], preferably the composition is a pharmaceutical composition.
[0216] 27. A method of treating cancer or a viral infection comprising the
step of
administering the antigen binding protein of any one of the above [15] to
[25], or the composition
of [26], to a patient in need thereof.
[0217] 28. A bivalent bispecific antigen binding protein comprising:
a) a Fab domain which specifically binds to a cell surface protein of an
immune
cell; and
b) at least two peptide-MFIC (pMHC) binding domains targeting the same pMHC
complex, wherein
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(i) one of the at least two pMEIC binding domains is operably linked to the C-
terminus of the heavy chain, and the other pMFIC binding domain is operably
linked to the C-terminus of the light chain, or
(ii) one of the at least two pMFIC binding domains is operably linked to the C-
terminus of the heavy chain, and the other pMFIC binding domain is operably
linked to the N-terminus of the light chain;
wherein the at least first and/or the at least second pMHC binding domain is
each
any one of a scFv, a scFab, a diabody, a sdAb (VI-ITI) or a Fab; and
wherein said bivalent bispecific antigen binding protein:
triggers or provides for MEC-restricted T cell activation, as described
elsewhere
herein; and/or
induces immune cell-mediated cytotoxicity towards a cell comprising the pMFIC
complex with higher potency as compared to a corresponding monovalent
bispecific antigen binding protein targeting a single pMEIC complex, as
determined under the same conditions. In some embodiments, said cell
comprising
the pMFIC complex is a cancer cell. T cell activation may, e.g., be determined
by
IFN-y (gamma) release, or may be determined by quantification of CD69 and
CD25 markers on CD8+ T cell populations after 24h using flow cytometry, as
exemplified in the Examples.
[0218] 29. The bivalent bispecific antigen binding protein of [28], wherein
the at least first
target plVIEIC complex and the at least second target plVfHC complex are the
same or are different.
Preferably, they are the same.
[0219] 30. The bivalent bispecific antigen binding protein of [28] or [29],
comprising at
least 5 amino acids of an antibody hinge region, located at the C-terminus of
the CHI domain of
the Fab domain which specifically binds to a cell surface protein of an immune
cell.
[0220] 31. In any one of the above [28] to [30], the Fab domain which
specifically binds
to a cell surface protein of an immune cell comprises a variable heavy chain
having a non-polar
amino acid at position 11, 89 and/or 108, according to Kabat numbering, as
described elsewhere
herein.
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[0221] 32. In any one of the above [28] to [31], the at least two pMEIC
binding domains
comprise a variable heavy chain having
(i) a polar amino acid as described elsewhere herein, e.g., serine, at
position 11, 89
and/or 108, according to Kabat numbering, as described elsewhere herein;
and/or
(ii) a deletion or substitution at position 112 and/or position 113, as
described
elsewhere herein.
[0222] 33. The bivalent bispecific antigen binding protein of any of the above
[28] to [32],
wherein the at least two pMHC binding domains specifically target an MIIC
restricted peptide
derived of a tumor antigen or a viral antigen.
[0223] 34. The bivalent bispecific antigen binding protein of
any one of the above
[28] to [33], wherein the Fab domain specifically binds to CD3 with a binding
affinity (KD)
between about 1 nM to about 50 nM, as determined by SPR.
[0224] 35. The bivalent bispecific antigen binding protein of any one of the
above [28] to
[34], wherein the at least two pMEIC binding domains bind the target peptide
pMlIC complex
with a binding affinity (Ku) of about 100 pM to about 20 nM. In preferred
embodiments, the at
least first pMIIC binding domain and/or the at least second pMEIC binding
domain binds the
target peptide pMEIC complex with a binding affinity (KD) of about 500 pM to
about 10 n1V1 or
of about 500 pM to about 5 nM.
[0225] 36. The bivalent bispecific antigen binding protein of any one of the
above [28] to
[35], comprising a molecular weight of about 75 kDa to about 110 kDa.
[0226] 37. The bivalent bispecific antigen binding protein of any one of the
above [28] to
[36], wherein the bivalent bispecific antigen binding protein has increased
serum half-life relative
to a bivalent bispecific antigen binding protein with a molecular weight of
less than about 60 kDa.
[0227] 38. A composition comprising the bivalent bispecific antigen binding
protein of
any one of the above [28] to [36], preferably the composition is a
pharmaceutical composition.
[0228] 39. A method of treating cancer or a viral infection comprising the
step of
administering the bivalent bispecific antigen binding protein of any one of
the above [28] to [37],
or the composition of [38], to a patient in need thereof.
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[0229] 40. A bispecific T cell engager which is bivalent for a pMHC target,
wherein said
bivalent T cell engager exhibits increased pMHC cell expressing toxicity than
a corresponding
monovalent bispecific.
[0230] 41. The bispecific T cell engager of [40], having the structural and
functional
features as described elsewhere herein, e.g., in [1]-[14] or in [15]-[25].
[0231] 42. A bispecific T cell engager, comprising a CD3 binding domain and at
least one
pMHC binding domain, preferably two pMHC complex binding domains, wherein the
association
rate constant ka and/or the dissociation rate constant kd of the CD3 binding
domain similar for
both CD3-heterodimers CD3Ey (epsilon/gamma) and CD3E6 (epsilon/delta) when
determined by
SPR at 25 C.
[0232] 43. The bispecific T cell engager of [42], having the structural and
functional
features as described elsewhere herein, e.g., in [1]-[14] or in [15]-[25].
[0233] 44. The bispecific T cell engager of [42] or [43], wherein the
association rate
constant ka of the CD3 binding domain is between about lx 105 to about 1 x107
M's', the
dissociation rate constant kd of the CD3 binding domain is between about I
x104 to about 1 10-
6 the association rate constant, ka of the pMHC binding domain is
between about lx 105 to
about 1 x 107 M-ls-1 and the dissociation rate constant kd of the pMHC binding
domain is between
about lx 104 to about 1 x 10-6
[0234] 45. The bispecific T cell engager of any of the above [44]- [42],
wherein the
association rate constant ka and/or the dissociation rate constant kd of the
CD3 binding domain are
similar to the association rate constant ka and/or the dissociation rate
constant kd of the pMEIC
binding domains.
Peptide-MEC Complex binding domains
[0235] As is known in the art, ME1C molecules present peptides, in particular
antigenic
peptides, on the surface of cells to be recognized by immune cells.
Accordingly, as will be
appreciated by a skilled artisan, the term "pMHC complex" as used herein
refers to a complex of
an MFIC molecule and a peptide, in particular an antigenic peptide, presented
by the MEC
molecule. This is commonly known as MTC-restricted antigen presentation.
Accordingly, the
peptide targeted by the pMHC binding domains is an MEC-restricted peptide. The
peptide can
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thus be considered as target peptide or target antigenic peptide. Further, in
accordance with the
present disclosure, the terms "target pMHC binding domain" and "pMHC binding
domain" may
be used interchangeably herein, and in any case refer to the at least first
and at least second pMHC
binding domains referred to herein throughout. The terms "target
peptide/antigen presented by a
MEC molecule/complex" and "MHC restricted target peptide/antigen", or similar
expressions
used throughout the present specification, may be used interchangeably herein.
[0236] While MHC occur in all vertebrates, the MHC in human is known as HLA
(human
leukocyte antigen). There are three classes of MHC molecules. The target
peptide may be
presented on a MILIC class I complex (such as of serotype HLA-A, HLA-B, HLA-C,
HLA-E,
HLA-F, HLA-G, HLA-K or HLA-L, or their respective subtypes) or an MHC class II
complex
(such as the serotypes HLA-DP, HLA-DQ, HLA-DR, DM or DO, or their respective
subtypes).
Each of the serotypes comprise different subtypes. In one embodiment, the
antigen binding protein
targets a peptide bound to an HLA-A2 -MI-IC complex, also termed HLA-A*02, in
particular
HLA-A*02.01
[0237] In certain embodiments, the antigen binding protein selectively binds a
pMHC
complex of a given HLA subtype and a target peptide, but not to a pMHC complex
of the same
HLA subtype presenting a different peptide. In certain embodiments, the
antigen binding protein
selectively binds to a target peptide presented on a pMHC complex of a given
HLA subtype, but
not to the same of peptide presented on a pMHC complex of a different HLA
subtype.
[0238] Accordingly, the present disclosure encompasses, in certain
embodiments, a
bispecific bivalent antigen binding protein, comprising a Fab domain which
specifically binds to
CD3, and no more than two pMHC binding domains, wherein both pMHC binding
domains are
targeting the same HLA-A complex (i.e., the antigen binding protein is
bivalent with regard to the
target pMHC complex), such as the same HLA-A2 complex, or are targeting the
same peptide
presented by a HLA-A complex, in particular presented by a HLA-A2 complex,
wherein both
pMHC binding domains are each a scFv, or are each a sdAb (VIM), and wherein
(i) one of the
two pMHC binding domains is operably linked to the C-terminus of the heavy
chain of the CD3
binding domain, and the other pMHC binding domain is operably linked to the C-
terminus of the
light chain of the CD3 binding domain, or (ii) one of the two pMHC binding
domains is operably
linked to the C-terminus of the heavy chain of the CD3 binding domain, and the
other pMHC
binding domain is operably linked to the N-terminus of the light chain of the
CD3 binding domain.
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[0239] In certain embodiments, the MHC restricted peptide is derived from a
tumor
antigen or a viral antigen. In some embodiments, the MEW restricted peptide is
a cancer testis
antigen. In some embodiments, the MHC restricted peptide is a neoantigen. In
certain
embodiments, the MHC restricted peptide is derived from a NY-ESO-1 (New York
esophageal
squamous cell carcinoma-1) protein, PRAME (preferentially expressed antigen in
melanoma)
protein or SX-2 (Synovial Sarcoma, X breakpoint 2) protein.
[0240] In certain embodiments, the MHC restricted target peptide is derived
from a
MAGE protein, including the MAGE-A, -B, -C subfamily members. In some
embodiments, the
MHC restricted target peptide is derived from a MAGE-A protein, including but
not limited to
MAGE-AL MAGE-A2, MAGE-A3, and MAGE-A4. In some embodiments, the MEW restricted
target peptide is derived from a MAGE-A4 protein. As described elsewhere
herein and as
exemplified by the Examples and the drawings, in preferred embodiments, the
target peptide is
presented on an MHC class I molecule of serotype HLA-A, preferably HLA-A2,
wherein the
target peptide derived from a MAGE-A protein, preferably from a MAGE-A4
protein., thus
certain antigen binding proteins described herein possess binding specificity
to a MAGE-A4
peptide-MHC.
[0241] In one embodiment, the target peptide is GVYDGREHTV (SEQ ID NO.: 1)
which
corresponds to amino acids 230-239 of MAGE-A4. Thus, in one embodiment, a
bivalent antigen
binding protein of the present disclosure may have binding affinity (Ku) to
the target peptide
GVYDGREHTV (SEQ ID NO.: 1); and/or may trigger or provide for 'VLF-IC-
restricted T cell
activation, as described elsewhere herein. T cell activation may, e.g., be
determined by IFN-7
(gamma) release, or may be determined by quantification of CD69 and CD25
markers on CD8
T cell populations after 24h using flow cytometry, as exemplified in the
Examples.
[0242] As described elsewhere herein and as exemplified in the Examples and
the
drawings, in some embodiments thereof, the at least first and at least second
pMHC binding
domain are the same and are in particular each an scFv and are the same, or
are each a sdAb and
are the same. Further, as described elsewhere herein and as exemplified in the
Examples and the
drawings, in some embodiments thereof, the target peptide is presented on an
MHC class I
molecule of serotype HLA-A2. In some embodiments thereof, the target
(antigenic) peptide (or
MHC restricted target peptide) is derived from a MAGE protein, preferably from
a MAGE-A4
protein. As further described elsewhere herein and as further exemplified in
the Examples and the
drawings, in some embodiments, the antigen binding protein has no more than
two pMFIC binding
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domains, i.e., is limited with regard to pMHC binding domains to one first
plVIEIC binding domain
one second pMHC binding domain, which preferably are both an scFy and are the
same, or which
are both a sdAb and are the same.
[0243] Accordingly, in various embodiments, an antigen binding protein as
provided by
the present disclosure comprises at least a first pMHC binding domain and at
least a second pMHC
binding domain, each binding to a first or second pMHC complex presenting the
target peptide
GVYDGREHTV (SEQ ID NO.: 1). According to some embodiments described elsewhere
herein,
and as exemplified in the Examples and the drawings, an antigen binding
protein as provided by
the present disclosure is bivalent for the pMEIC complex and comprises no more
than two pMHC
binding domains, wherein both pMHC binding domains binds to a pMHC complex
presenting the
target peptide GVYDGREHTV (SEQ ID NO.: 1). In some embodiments thereof, said
bivalent
antigen binding protein is bispecific, i.e., further to the binding
specificity for the target peptide
GVYDGREHTV (SEQ ID NO.: 1), it has binding specificity for a cell surface
protein of an
immune cell as described elsewhere herein, such CD3 Furthermore, in certain
embodiments
thereof, the pMHC complex presenting the target peptide of SEQ ID NO.: 1 is a
pMHC class I
complex, i.e., the target peptide is presented on an MEW class I molecule,
particularly on an MTIC
class I molecule of serotype HLA-A ("HLA-A pMHC complex"), more particularly
on an MHC
class I molecule of serotype HLA-A2 ("HLA-A2 pMHC complex-), as described
elsewhere
herein. As described elsewhere herein, and as exemplified by the Examples and
the drawings, in
some embodiments thereof, both pMHC binding domains are each a scFv, or are
each a sdAb
(VHH). As described elsewhere herein, and as exemplified by the Examples and
the drawings, in
further embodiments thereof, (i) one of the two pMHC binding domains is
operably linked to the
C-terminus of the heavy chain of the CD3 binding domain, and the other pMHC
binding domain
is operably linked to the C-terminus of the light chain of the CD3 binding
domain, or (ii) one of
the two pMHC binding domains is operably linked to the C-terminus of the heavy
chain of the
CD3 binding domain, and the other pMHC binding domain is operably linked to
the N-terminus
of the light chain of the CD3 binding domain.
[0244] The present disclosure encompasses antigen binding proteins comprising
scFvs
and sdAbs as described in W02022190009A1, which is hereby incorporated by
reference.
Accordingly, in various embodiments, an antigen binding protein as provided by
the present
disclosure comprises at least a first pMHC binding domain and at least a
second pMHC binding
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domain, wherein at least one of the at least first and at least second pMHC
binding domains
comprises:
(a) a heavy chain variable (VH) domain comprising
(i) an HCDR1 amino acid sequence of SNYAMS (SEQ ID NO.: 26),
(ii) an HCDR2 amino acid sequence of IVSSGGTTYYAX1X2X3KG (SEQ ID NO.. 27),
wherein Xi corresponds to amino acid S or D, X2 corresponds to amino acid W or
S, and
X3 corresponds to amino acid A or V, and
(iii) an HCDR3 amino acid sequence of DLYYGPX4TX5YX6X7X8NL (SEQ ID NO.: 28),
wherein X4 corresponds to amino acid T, N, or S, X5 corresponds to amino acid
D or is
absent, X6 corresponds to amino acid S or F, X7 corresponds to amino acid A or
V. and
X8 corresponds to amino acid F or A; and
(b) a light chain variable (VL) domain comprising
(iv) an LCDR1 amino acid sequence of TADTLSRSYAS (SEQ ID NO.: 29),
(v) an LCDR2 amino acid sequence of RDTSRPS (SEQ ID NO.. 30), and
(vi) an LCDR3 amino acid sequence of ATX9XioXiiSGSNFQX12 (SEQ ID NO.: 31),
wherein X9 corresponds to amino acid S or R, Xio corresponds to amino acid D
or P. Xi
corresponds to amino acid G, S, or F, and X12 corresponds to amino acid L or
A,
Wherein the antigen binding protein (in particular the at least first and/or
second pMTIC
binding domain) has binding affinity (Ku) to a WIC complex presenting the
target peptide
GVYDGREHTV (SEQ ID NO.: 1); and/or
wherein the antigen binding protein triggers or provides for MTIC-restricted T
cell
activation. T cell activation may, e.g., be determined by IFN-7 (gamma)
release, or may
be determined by quantification of CD69 and CD25 markers on CD8 T cell
populations
after 24h using flow cytometry, as exemplified in the Examples.
[0245] In certain embodiments of the disclosure, the antigen binding protein
exhibits a
specific binding affinity (Ku) to the MT1C presented target peptide GVYDGREHTV
(SEQ ID
NO.: 1) in the low nanomolar and/or even picomolar range, as described
elsewhere herein.
[0246] According to certain embodiments described elsewhere herein, and as
exemplified
in the Examples and the drawings, an antigen binding protein as provided by
the present disclosure
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is bivalent for p1V1TIC complex binding and comprises no more than two plVIEIC
binding domains,
wherein both pMHC binding domains comprise a VH and a VL domain as described
above (i.e.,
comprising the CDRs of SEQ ID NOs: 26-31), wherein said antigen binding
protein specifically
binds to a MHC complex presenting GVYDGREHTV (SEQ ID NO.: 1), in particular
HLA-A2
restricted GVYDGREHTV (SEQ ID NO.: 1), as described above; and/or wherein the
antigen
binding protein triggers or provides for 1V1TIC-restricted T cell activation
as described above. In
certain embodiments thereof, the bivalent antigen binding protein is
bispecific and has binding
specificity for a cell surface protein of an immune cell as described
elsewhere herein, such as
CD3. As described elsewhere herein, and as exemplified by the Examples and the
drawings, in
certain embodiments thereof, both said pMTIC binding domains are each a scFv,
or are each a
sdAb (VHH). As described elsewhere herein, and as exemplified by the Examples
and the
drawings, in further embodiments thereof, (i) one of the two pMHC binding
domains is operably
linked to the C-terminus of the heavy chain of the CD3 binding domain, and the
other pMHC
binding domain is operably linked to the C-terminus of the light chain of the
CD3 binding domain,
or (ii) one of the two pMHC binding domains is operably linked to the C-
terminus of the heavy
chain of the CD3 binding domain, and the other pMHC binding domain is operably
linked to the
N-terminus of the light chain of the CD3 binding domain.
[0247] In one embodiment, the pMHC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 2, 8, 14 or 20.
[0248] In one embodiment, the pMHC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR2 sequence of SEQ ID NO.: 3, 9, 15 or 21.
[0249] In one embodiment, the pMHC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR3 sequence of SEQ ID NO.: 4, 10, 16 or 22.
[0250] In one embodiment, the pMHC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR1 sequence of SEQ ID NO.: 5, 11, 17 or 23.
[0251] In one embodiment, the pMHC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR2 sequence of SEQ ID NO.: 6, 12, 18 or 24.
[0252] In one embodiment, the pMHC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR3 sequence of SEQ ID NO.: 7, 13, 19 or 25.
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[0253] In one embodiment, the pMEIC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 2, a HCDR2 sequence of
SEQ ID
NO.: 3, and a HCDR3 sequence of SEQ ID NO.: 4.
[0254] In one embodiment, the pMHC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 8, a HCDR2 sequence of
SEQ ID
NO.: 9, and a HCDR3 sequence of SEQ ID NO.: 10.
[0255] In one embodiment, the pMFIC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 14, a HCDR2 sequence of
SEQ ID
NO.: 15, and a HCDR3 sequence of SEQ ID NO.. 16.
[0256] In one embodiment, the pMEIC binding domain comprises a heavy chain
variable
(VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 20, a HCDR2 sequence of
SEQ ID
NO.: 21, and a HCDR3 sequence of SEQ ID NO.: 22.
[0257] In one embodiment, the pIVIHC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR1 sequence of SEQ ID NO.: 5, a LCDR2 sequence of
SEQ ID
NO.: 6, and a LCDR3 sequence of SEQ ID NO.: 7.
[0258] In one embodiment, the pMEIC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR1 sequence of SEQ ID NO.: 11, a LCDR2 sequence of
SEQ ID
NO.: 12, and a LCDR3 sequence of SEQ ID NO.: 13.
[0259] In one embodiment, the pl\THC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR1 sequence of SEQ ID NO.: 17, a LCDR2 sequence of
SEQ ID
NO.: 18, and a LCDR3 sequence of SEQ ID NO.: 19.
[0260] In one embodiment, the pMFIC binding domain comprises a light chain
variable
(VL) domain comprising a LCDR1 sequence of SEQ ID NO.: 23, a LCDR2 sequence of
SEQ ID
NO.: 24, and a LCDR3 sequence of SEQ ID NO.: 25.
[0261] In one embodiment, the pMEIC binding domain comprises (a) a heavy chain
variable (VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 2, a HCDR2
sequence of
SEQ ID NO.: 3, and a HCDR3 sequence of SEQ ID NO.: 4 and (b) a light chain
variable (VL)
domain comprising a LCDR1 sequence of SEQ ID NO.: 5, a LCDR2 sequence of SEQ
ID NO.:
6, and a LCDR3 sequence of SEQ ID NO.: 7.
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[0262] In one embodiment, the plVIEIC binding domain comprises (a) a heavy
chain
variable (VH) domain comprising a HCDRI sequence of SEQ ID NO.: 8, a HCDR2
sequence of
SEQ ID NO.: 9, and a HCDR3 sequence of SEQ ID NO.: 10 and (b) a light chain
variable (VL)
domain comprising a LCDR1 sequence of SEQ ID NO.: 11, a LCDR2 sequence of SEQ
ID NO.:
12, and a LCDR3 sequence of SEQ ID NO.: 13.
[0263] In one embodiment, the pMTIC binding domain comprises (a) a heavy chain
variable (VH) domain comprising a HCDR1 sequence of SEQ ID NO.: 14, a HCDR2
sequence
of SEQ ID NO.: 15, and a HCDR3 sequence of SEQ ID NO.: 16 and (b) a light
chain variable
(VL) domain comprising a LCDRI sequence of SEQ ID NO.: 17, a LCDR2 sequence of
SEQ ID
NO.: 18, and a LCDR3 sequence of SEQ ID NO.: 19.
[0264] In one embodiment, the pMTIC binding domain comprises (a) a heavy chain
variable (VH) domain comprising a HCDRI sequence of SEQ ID NO.: 20, a HCDR2
sequence
of SEQ ID NO.: 21, and a HCDR3 sequence of SEQ ID NO.: 22 and (b) a light
chain variable
(VL) domain comprising a LCDRI sequence of SEQ ID NO.: 23, a LCDR2 sequence of
SEQ ID
NO.: 24, and a LCDR3 sequence of SEQ ID NO.: 25.
[0265] Accordingly, in various embodiments, an antigen binding protein as
provided by
the present disclosure comprises at least a first pMHC binding domain and at
least a second plVIEIC
binding domain, wherein at least one of the at least first and at least second
pMHC binding
domains comprises the CDR sequences of any one of (i) SEQ ID NOs: 2-7, (ii)
SEQ ID NOs: 8-
13, (iii) SEQ ID NOs: 14-19, or (iv) SEQ ID NOs: 20-25, wherein the antigen
binding protein (in
particular the at least first and/or second pMHC binding domain) has binding
specificity to a MTIC
complex presenting the target peptide GVYDGREHTV (SEQ ID NO.: 1) as described
above in
the context of SEQ ID NOs: 26-31; and/or wherein the antigen binding protein
triggers or provides
for MHC-restricted T cell activation as described above in the context of SEQ
ID NOs: 26-31.
[0266] According to various embodiments described elsewhere herein, and as
exemplified
in the Examples and the drawings, an antigen binding protein as provided by
the present disclosure
is bivalent for the pMHC complex and comprises no more than two pIVITIC
binding domains,
wherein both pMHC binding domains comprise a VH and a VL domain as described
above (i.e.,
comprising the CDRs of any one of (i) SEQ ID NOs: 2-7, (ii) SEQ ID NOs: 8-13,
(iii) SEQ ID
NOs: 14-19, or (iv) SEQ ID NOs: 20-25), wherein the antigen binding protein
(in particular the
two pMHC binding domains) has binding affinity (Kr) to a MHC complex
presenting the target
peptide GVYDGREHTV (SEQ ID NO.: 1), in particular HLA-A2 restricted, as
described above;
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and/or wherein the antigen binding protein triggers or provides for MHC-
restricted immune cell
activation as described above. In certain embodiments thereof, the bivalent
antigen binding
protein is bispecific and has binding specificity for CD3 as described
elsewhere herein. More
particularly, the immune cell binding domain of said antigen binding protein
which specifically
binds to CD3 is monovalent for CD3 and may be a Fab. As described elsewhere
herein, and as
exemplified by the Examples and the drawings, in some embodiments thereof,
both pMTIC
binding domains are each a scFv, or are each a sdAb (VHH). As described
elsewhere herein, and
as exemplified by the Examples and the drawings, in further embodiments
thereof, (i) one of the
two pMHC binding domains is operably linked to the C-terminus of the heavy
chain of the CD3
Fab domain, and the other pMI-IC binding domain is operably linked to the C-
terminus of the light
chain of the CD3 binding Fab domain, or (ii) one of the two pMFIC binding
domains is operably
linked to the C-terminus of the heavy chain of the CD3 binding Fab domain, and
the other pMTIC
binding domain is operably linked to the N-terminus of the light chain of the
CD3 binding Fab
domain.
[0267] In certain embodiments, the CDRs are derived from an antigen binding
protein
disclosed herein, such as those disclosed in Tables 2, 3 or 4.
[0268] Table 2 ¨ CDR sequences of exemplary pMHC binding domains targeting the
peptide of SEQ ID NO.:1 presented by HLA-A*02:01
SEQ ID NO.: 2 SNYAM S
(M1 HCDR1)
SEQ ID NO.: 3 IVSSGGTTYYADSVKG
(M1 HCDR2)
SEQ ID NO 4 DLYYGPNTDYSAANL
(M1 HCDR3)
SEQ ID NO.: 5 TADTLSRSYAS
(M1 LCDR1)
SEQ ID NO.: 6 RDTSRPS
(M1 LCDR2)
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SEQ ID NO.: 7 ATRPSSGSNFQA
(M1 LCDR3)
SEQ ID NO.: 8 SNYAMS
(M2 HCDR1)
SEQ ID NO 9 IVSSGGTTYYADSVKG
(M2 HCDR2)
SEQ ID NO.. 10 DLYYGPSTYFVANL
(M2 HCDR3)
SEQ ID NO.: 11 TADTLSRSYAS
(M2 LCDR1)
SEQ ID NO.: 12 RDTSRPS
(M2 LCDR2)
SEQ ID NO 13 ATRPSSGSNFQL
(M2 LCDR3)
SEQ ID NO.. 14 SNYAMS
(M3 HCDR1)
SEQ ID NO.: 15 IVSSGGTTYYASWAKG
(M3 HCDR2)
SEQ ID NO.: 16 DLYYGPTTYSAANL
(M3 HCDR3)
SEQ ID NO 17 TADTLSRSYAS
(M3 LCDR1)
SEQ ID NO.. 18 RDTSRPS
(M3 LCDR2)
SEQ ID NO.. 19 ATRDFSGSNFQL
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(M3 LCDR3)
SEQ ID NO.. 20 SNYAMS
(M4 HCDRI)
SEQ ID NO.: 21 IVSSGGTTYYASWAKG
(M4 FICDR2)
SEQ ID NO.: 22 DLYYGPTTYSAFNL
(M4 HCDR3)
SEQ ID NO.: 23 TADTLSRSYAS
(M4 LCDR1)
SEQ ID NO.. 24 RDTSRPS
(M4 LCDR2)
SEQ ID NO.: 25 ATRPSSGSNFQA
(M4 LCDR3)
SEQ ID NO.: 26 SNYAMS
Consensus
HCDRI
SEQ ID NO.: 27 IVSSGGTTYYAX1X2X3KG
Consensus wherein Xi corresponds to amino acid S or D, X2
corresponds to
HCDR2 amino acid W or S, and X3 corresponds to amino
acid A or V
SEQ ID NO.: 28 DLYYGPX4TX5YX6X7X8NL
Consensus wherein X4 corresponds to amino acid T, N, or
S, X5 corresponds
HCDR3 to amino acid D or is absent, X6 corresponds to
amino acid S or F,
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X7 corresponds to amino acid A or V. and X8 corresponds to amino
acid F or A
SEQ ID NO.: 29 TADTLSRSYAS
Consensus
LCDR1
SEQ ID NO.: 30 RDTSRPS
Consensus
LCDR2
SEQ ID NO.: 31 ATX9X10X11SGSNFQX12
Consensus wherein X9 corresponds to amino acid S or R,
Xio corresponds to
LCDR3 amino acid D or P, Xii corresponds to amino
acid G, S, or F, and
X12 corresponds to amino acid L or A.
[0269] In one embodiment, the antigen binding protein comprises an amino acid
sequence
of SEQ ID NOs: 32, 34, 36 or 38. In one embodiment, the antigen binding
protein comprises an
amino acid sequence of SEQ ID NOs: 33, 35, 37 or 39. In one embodiment, the
antigen binding
protein comprises the amino acid sequences of SEQ ID NOs: 32 and 33. In one
embodiment, the
antigen binding protein comprises the amino acid sequences of SEQ ID NOs: 34
and 35 In one
embodiment, the antigen binding protein comprises the amino acid sequences of
SEQ ID NOs:
36 and 37. In one embodiment, the antigen binding protein comprises the amino
acid sequences
of SEQ ID NOs: 38 and 39.
[0270] Also encompassed are variants of the sequences disclosed herein. A
variant amino
acid or nucleic acid sequence differs from its parental sequence by virtue of
insertion (including
addition), deletion and/or substitution of one or more amino acid residues or
nucleobases,
respectively, while retaining at least one desired activity of the parent
sequence disclosed herein,
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e.g., specific antigen binding. Variants may be artificially engineered or
naturally occurring, such
as e.g., allelic or splice variants.
[0271] Thus, in certain embodiments, a variant antigen binding protein retains
specific
binding to its target (e.g., an HLA-A2 restricted GVYDGREHTV, SEQ ID NO.: 1)
and/or
competes with an antigen binding protein disclosed herein for binding to its
target. In certain
embodiments, the variant antigen binding protein comprises an amino acid
sequence being at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence
disclosed herein. In certain embodiments, the variant antigen binding protein
comprises 1, 2, 3_4
,5 ,6 ,7, 8, 9, or 10 substitutions with regard to the parental amino acid
sequence.
[0272] Table 3 ¨ Heavy and light chain amino acid sequences of exemplary pMHC
domains. CDR sequences are highlighted in bold underlined text.
SEQ ID NO.: 32 EVQLLESGGGSVQPGGSLRLSCTVSGFSLSNYAMSWVRQ
APGKGLEWIGIVS SGGTTYYA D SVKGRF TISRDNSKNTVY
LQMNSLRAEDTASYYCAKDLYYGPNTDYSAANLWGQGT
SVTVSS
SEQ ID NO.: 33 QSVLTQDPAVSVALGQTVRITCTADTLSRSYASWYQQKP
GQAPVLVIYRDTSRPSGIPDRFSGSSSGNTASLTITGAQAE
DEADYYCATRPSSGSNFQAFGGGTKLTVLG
SEQ ID NO.: 34 EVQLLESGGGSVQPGGSLRLSCTVSGFSLSNYAMSWVRQ
APGKCLEWIGIVSSGGTTYYADSVKGRFTISRDNSKNTVY
LQMNSLRAEDTASYYCAKDLYYGPSTYFVANLWGQGTS
VTVSS
SEQ ID NO.: 35 QSVLTQDPAVSVALGQTVRITCTADTLSRSYASWYQQKP
GQAPVLVIYRDTSRPSGIPDRFSGSSSGNTASLTITGAQAE
DEADYYCATRPSSGSNFOLFGCGTKLTVLG
SEQ ID NO.: 36 EVQLLESGGGSVQPGGSLRLSCTVSGFSLSNYAMSWVRQ
APGKCLEWIGIVSSGGTTYYASWAKGRFTISKDT SKNTV
YLQMNSLRAEDTASYYCAKDLYYGPTTYSAANLWGQGT
SVTVSS
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SEQ ID NO.: 37 SYELTQPPSVSVSPGQTASITCTADTLSRSYASWYQQKPG
QSPVLVIYRDTSRPSGIPERF S GSN S GNTATLTIS GT QAMD
EADYYCATRDFSGSNFOLFGCGTKLTVLG
SEQ ID NO.: 38 EVQLLE S GGGS VQPGGSLRL SC TVS GF SLSNYAMSWVRQ
APGKGLEYIGIVSSGGTTYYASWAKGRFTISRDNSKNTVY
LQMNSLRAEDTASYYCAKDLYYGPTTYSAFNLWGQGTS
VTVSS
SEQ ID NO.: 39 SYELTQPPSVSVSPGQTASITCTADTLSRSYASWYQQKPG
QSPVLVIYRDTSRPSGIPERF S GSN S GNTATLTIS GT QAMD
EADYYCATRPSSGSNFQAF GGGTKLTVLG
[0273] Accordingly, in various embodiments, an antigen binding protein as
provided by
the present disclosure comprises at least a first pMI-IC binding domain and at
least a second pMFIC
binding domain, wherein at least one of the at least first and at least second
pMEIC binding
domains comprises the VH/VL sequences of any one of (i) SEQ ID NOs: 32-33,
(ii) SEQ ID NOs:
34-35, (iii) SEQ ID NOs: 36-37, or (iv) SEQ ID NOs: 38-39, wherein the antigen
binding protein
(in particular the at least first and/or second pMHC binding domain) has
binding affinity (KD) to
the target peptide GVYDGREHTV (SEQ ID NO.: 1) as described above in the
context of SEQ
ID NOs: 26-31; and/or wherein the antigen binding protein triggers or provides
for MTIC-
restricted T cell activation as described above in the context of SEQ ID NOs:
26-31.
[0274] According to preferred embodiments described elsewhere herein, and as
exemplified in the Examples and the drawings, an antigen binding protein as
provided by the
present disclosure is bivalent for the pMFIC complex and comprises no more
than two pIVIIIC
binding domains, wherein both p1M-FIC binding domains comprise a VH and a VL
domain as
described above (i.e., comprising the VH/VL sequences of any one of (i) SEQ ID
NOs: 32-33, (ii)
SEQ ID NOs: 34-35, (iii) SEQ ID NOs: 36-37, or (iv) SEQ ID NOs: 38-39),
wherein the antigen
binding protein (in particular the two pMHC binding domains) specifically
binds to the target
pMFIC presenting GVYDGREHTV (SEQ ID NO.: 1), in particular HLA-A2 restricted,
as
described above; and/or wherein the antigen binding protein triggers or
provides for MTIC-
restricted "I cell activation as described above. In preferred embodiments,
the bivalent antigen
binding protein is bispecific and has binding specificity for a cell surface
protein of an immune
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cell as described elsewhere herein, such as binding specificity for CD3 as
described elsewhere
herein. In some embodiments thereof, the immune cell binding domain is a Fab
domain which
specifically binds to CD3. As described elsewhere herein, and as exemplified
by the Examples
and the drawings, in some embodiments thereof, both pIVIEIC binding domains
are each a scFv,
or are each a sdAb (VHH). As described elsewhere herein, and as exemplified by
the Examples
and the drawings, in further embodiments thereof, (i) one of the two plVIFIC
binding domains is
operably linked to the C-terminus of the heavy chain of the CD3 binding Fab
domain, and the
other plVIIIC binding domain is operably linked to the C-terminus of the light
chain of the CD3
binding Fab domain, or (ii) one of the two pMTIC binding domains is operably
linked to the C-
terminus of the heavy chain of the CD3 binding (Fab) binding domain, and the
other plVIEIC
binding domain is operably linked to the N-terminus of the light chain of the
CD3 binding Fab
domain.
[0275] Accordingly, the present disclosure encompasses, in certain
embodiments, a
bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the HCDR sequences of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ
ID NOs.:
79, 81 and 82 or variants thereof; and no more than two pMTIC binding domains
targeting the
same pMHC complex, wherein both pMEIC binding domains are each a scFv, said
scFvs
comprising the CDRs of SEQ ID NOs: 26-31, the bispecific bivalent antigen
binding protein e.g.,
being a Fab-(scFv)2. Accordingly, the present disclosure encompasses, in
certain embodiments, a
bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the HCDR sequences of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ
ID NOs.:
80, 81 and 82 or variants thereof; and no more than two pMTIC binding domains
targeting the
same plVIFIC complex, wherein both pIVEFIC binding domains are each a scFv,
said scFvs
comprising the CDRs of SEQ ID NOs: 26-31, the bispecific bivalent antigen
binding protein e.g.,
being a Fab-(scFv)2. Accordingly, the present disclosure encompasses, in
certain embodiments, a
bispecific bivalent antigen binding protein, comprising an anti-CD3-binding
domain comprising
the HCDR sequences of SEQ ID NOs.: 76, 77 and 78 and the LCDR sequences of SEQ
ID NOs.:
79, 81 and 82 or variants thereof; and no more than two pMEIC binding domains
targeting the
same pMHC complex, wherein both pIVITIC binding domains are each a scFv, said
scFvs
comprising the CDRs of (i) SEQ ID NOs: 2-7, (ii) SEQ ID NOs: 8-13, (iii) SEQ
ID NOs: 14-19,
or (iv) SEQ ID NOs: 20-25, or variants thereof, respectively, the bispecific
bivalent antigen
binding protein e.g., being a Fab-(scFv)2. Accordingly, the present disclosure
encompasses, in
certain embodiments, a bispecific bivalent antigen binding protein, comprising
an anti-CD3-
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binding domain comprising the HCDR sequences of SEQ ID NOs.: 76, 77 and 78 and
the LCDR
sequences of SEQ ID NOs.: 80, 81 and 82 or variants thereoff, and no more
than two pMTIC
binding domains targeting the same plVITIC complex, wherein both pMHC binding
domains are
each a scFv, said scFvs comprising the CDRs of (i) SEQ ID NOs: 2-7, (ii) SEQ
ID NOs: 8-13,
(iii) SEQ ID NOs: 14-19, or (iv) SEQ ID NOs: 20-25, or variants thereof,
respectively, the
bispecific bivalent antigen binding protein e.g., being a Fab-(scFv)2.
Accordingly, the present
disclosure encompasses, in certain embodiments, a bispecific bivalent antigen
binding protein,
comprising an anti-CD3-binding domain comprising the VL sequence of SEQ ID
NO.: 83 and the
VH sequence of SEQ ID NO.: 84 or variants thereof; and no more than two pMTIC
binding
domains targeting the same pMHC complex, wherein both pMEIC binding domains
are each a
scFv, said scFvs comprising the VH/VL sequences of any one of (i) SEQ ID NOs:
32-33, (ii) SEQ
ID NOs: 34-35, (iii) SEQ ID NOs: 36-37, or (iv) SEQ ID NOs: 38-39, or variants
thereof,
respectively, the bispecific bivalent antigen binding protein e.g., being a
Fab-(scFv)2. The
exemplary pMHC binding domains targeting the peptide of SEQ ID NO.: 1
presented by HLA-
A*02:01 and sequences recited above are described in further detail in U.S.
20220380472A1 and
U.S. Provisional Patent Application Serial No. 63/318,163, filed March 9,
2022, the contents of
each are incorporated herein by reference.
Reduction of Anti-Drug Antibody Binding
[0276] Anti-drug antibodies (ADAs) may affect the risk profile and efficacy of
a
biological drug. If neutralizing, they may block the drug's ability to bind to
its target. It is
therefore a regulatory requirement to test biologic drugs for the binding of
anti-drug antibodies
and their neutralizing potential. Anti-drug antibody assays are e.g., detailed
in
W02007101661A1 (Hoffmann La Roche), W02018178307A1 (Ablynx), W02021046316A2
(Adverum Biotechnologies, Charles River), and U520180088140A1 (Genzyme
Corporation),
each of which is incorporated herein by reference.
[0277] Anti-drug antibodies binding to a tumor targeting domain of an antigen
binding
protein may lead to clustering of said antigen binding protein when each
variable domain of the
ADA binds to one tumor targeting domain of two antigen binding proteins. The
two or more CD3
binding domains on said antigen binding protein cluster and overstimulate the
targeted T cell in
the absence of target engagement, thereby leading to off-target toxicity.
Unspecific stimulation of
the T-cells may lead to systemic cytokine release.
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[0278] Generally, there is a need in the art to develop safer and more
effective bispecific
antibodies for cancer immunotherapy.
[0279] The inventors have found that certain mutations in the tumor antigen
binding
domain of a T cell engager reduce ADA response and at the same time reduce
nonspecific T cell
stimulation in the absence of target engagement. Thereby, a highly effective
and safe approach
for cancer immunotherapy is provided.
[0280] In one aspect, the disclosure provides a method of reducing nonspecific
T cell
activation of a T cell engaging multispecific antigen binding protein, wherein
the multi specific
antigen binding protein comprises a first binding domain specifically
targeting CD3 and a second
binding domain specifically targeting a tumor antigen, wherein the
multispecific antigen binding
protein comprises at least one variable heavy chain, the method comprising the
steps of: a)
substituting a variable heavy chain amino acid at position 11, 89, and/or 108,
according to Kabat
numbering, with a polar amino acid; and b) deleting a serine (S) at position
113, according to
Kabat numbering.
[0281] In certain embodiments, the polar amino acid of step a) is serine (S)
and/or
threonine (T).
[0282] In certain embodiments, the heavy chain amino acid is substituted with
serine (S)
at heavy chain amino acid position 11, serine (S) or threonine (T) at heavy
chain amino acid
position 89, and/or serine (S) or threonine (T) at heavy chain amino acid
position 108, according
to Kabat numbering.
[0283] In certain embodiments, the heavy chain amino acid is substituted with
serine (S)
at heavy chain amino acid position 11, serine (S) at heavy chain amino acid
position 89, and serine
(S) at heavy chain amino acid position 108, according to Kabat numbering.
[0284] In certain embodiments, step b) further comprises the step of deleting
a serine (S)
at position 112, according to Kabat numbering.
[0285] In certain embodiments, the method further comprises adding alanine
(A), glycine
(G) or threonine (T) at Kabat amino position 112 or 113
[0286] In certain embodiments, the method further comprises adding alanine (A)
at Kabat
amino position 112 or 113.
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[0287] In certain embodiments, the multispecific antigen binding protein is
monovalent,
bivalent or multivalent.
[0288] In certain embodiments, the antigen binding protein of said method is a
Fab-sdAb,
Fab-(sdAb)2, a Fab-scFv or a Fab-(scFv)2, F(ab')2fragment, bis-scFv (or tandem
scFv or BiTE),
DART, diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc-scFab, IgG-seFv, scFv-Fc,
scFv-fc-scFv,
Fv2-Fc, FynomAB, quadroma, CrossMab, DuoBody, triabody and tetrabody, or
MATCH.
[0289] In certain embodiments, the second binding domain specifically targets
a p1V1I-IC.
[0290] In certain embodiments, the multispecific antigen binding protein
further
comprises a third binding domain specifically targeting a pMHC.
[0291] In certain embodiments, the second binding domain and the third binding
domain
specifically target the same pMHC or different plVIFIC
[0292] In certain embodiments, the antigen binding protein comprises one
binding domain
specifically targeting CD3 and one binding domain specifically targeting a pMI-
IC.
[0293] In certain embodiments, the antigen binding protein comprises one
binding domain
specifically targeting CD3 and two binding domains specifically targeting a
pMHC.
[0294] In certain embodiments, the two binding domains specifically targeting
a pMEC
are the same.
[0295] In certain embodiments, the pMHC binding domain specifically targets a
MEC
restricted peptide derived of a tumor antigen or a viral antigen.
[0296] In certain embodiments, the binding affinity (KD) for CD3 is between
about 1 nM
to about 50 nM, optionally between about 20 nM to 50 nM, as determined by SPR
[0297] In certain embodiments, the binding affinity (KD) for CD3 is of about 1
nM, of
about 10 nM, or of about 50 nM, as determined by SPR.
[0298] In certain embodiments, the binding affinity (KD) for CD3 is of about 1
nM, of
about 10 nM, or of about 50 nM, as determined by SPR.
[0299] In certain embodiments, the binding affinity (KD) for the pMHC is of
about 100
pM to about 20 nM (e.g., about 100 pM, about 150 pM, about 200 pM, about 250
pM, about 300
pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM,
about 600 pM,
about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about
900 pM, about
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950 pM, about 1 nM (1,000 pM), about 2 nM, about 3 nM, about 4 nM, or about 5
nM, about 6
nM, about 7 nM, about 8 n1\4, about 9 nM, about 10 nM, about 11 nM, about 12
nM, about 13
nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19
nM, or about
20 nM). In certain embodiments, the binding affinity (KD) for the pMEIC is of
about 100 pM to
about 10 nM. In certain embodiments, the binding affinity (KD) for the pMHC is
of about 500
pM to about 10 nM. In certain embodiments, the binding affinity (KD) for the
plVII-IC is of about
500 pM to about 5 n1\4. In certain embodiments, the binding affinity (KD) for
the pMHC is of
about 500 pM to about 2 nM In certain embodiments, the binding affinity (KD)
for the pMilIC is
500 pM to about 1 nM,
[0300] In another aspect, the disclosure provides a multispecific antigen
binding protein
obtainable by the methods described above.
[0301] In another aspect, the disclosure provides an antigen binding protein
comprising at
least one first binding domain specific for CD3 and at least one second
binding domain specific
for a tumor antigen, each binding domain comprising at least one variable
heavy chain, wherein
at least one variable heavy chain comprises a polar amino acid at position 11,
89 and/or 108,
according to Kabat numbering.
[0302] In certain embodiments, the variable heavy chain is of said second
binding domain.
[0303] In certain embodiments, the polar amino acid is serine (S) and/or
threonine (T).
[0304] In certain embodiments, the variable heavy chain comprises serine (S)
at heavy
chain amino acid position 11, serine (S) or threonine (T) at heavy chain amino
acid position 89,
and serine (S) or threonine (T) at heavy chain amino acid position 108,
according to Kabat
numbering.
[0305] In certain embodiments, the variable heavy chain comprises serine (S)
at heavy
chain amino acid position 11, serine (S) at heavy chain amino acid position
89, and serine (S) at
heavy chain amino acid position 108, according to Kabat numbering.
[0306] In certain embodiments, the variable heavy chain has a serine (S) at
position 113
deleted, according to Kabat numbering
[0307] In certain embodiments, the variable heavy chain has serine (S) at
position 112 and
113 deleted, according to Kabat numbering.
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[0308] In certain embodiments, the antigen binding protein comprises alanine
(A), glycine
(G) or threonine (T) at position 112, according to Kabat numbering, in
particular alanine (A).
[0309] In certain embodiments, the tumor antigen is a pMHC.
[0310] In certain embodiments, the pMHC binding domain specifically targets a
MTIC
restricted peptide derived of a tumor antigen or a viral antigen.
[0311] In certain embodiments, the antigen binding protein has an affinity
(Ku) for CD3
of about 1 nM to about 50 nM, optionally between about 20 nM to 50 nM, as
determined by SPR.
[0312] In certain embodiments, the antigen binding protein has an affinity
(Ku) for CD3
of about 1 nM, of about 10 nM or of about 50 nM, as determined by SPR.
[0313] In certain embodiments, the first binding domain specific for CD3 is a
Fab
fragment.
[0314] In certain embodiments, the antigen binding protein comprises two or
more pIVITIC
binding domains
[0315] In certain embodiments, the pMHC binding domain is a schi or an sdAb.
[0316] In certain embodiments, the antigen binding protein has an affinity
(Ku) for the
pMHC of about 100 pM to about 20 nM (e.g., about 100 pM, about 150 pM, about
200 pM, about
250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM,
about 550
pM, about 600 pM, about 650 pM, about 700 pM, about 750 pM, about 800 pM,
about 850 pM,
about 900 pM, about 950 pM, about 1 nM (1,000 pM), about 2 nM, about 3 nM,
about 4 nM,
about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about
11 nM, about
12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about
18 nM, about
19 nM, or about 20 nM) In certain embodiments, the antigen binding protein has
an affinity (KD)
for the pMFIC of about 100 pM to about 1 nM. In certain embodiments, the
antigen binding
protein has an affinity (Ku) for the pMHC of about 500 pM to about 2 nM. In
certain
embodiments, the antigen binding protein has an affinity (KD) for the pMHC of
about 500 pM to
about 3 nM. In certain embodiments, the antigen binding protein has an
affinity (KD) for the
pMHC of about 500 pM to about 5 nM.
[0317] In certain embodiments, said antigen binding protein is a Fab-sdAb, Fab-
(sdAb)2,
a Fab-scFv or a Fab-(scFv)2, F(ab1)2fragment, bis-scFv (or tandem scFv or
BiTE), DART,
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diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc-scFab, IgG-scFv, scFv-Fc, scFv-fc-
scFv, Fv2-Fc,
FynomAB, quadroma, CrossMab, DuoBody, triabody and tetrabody, or MATCH.
Expression of Antigen Binding Proteins
[0318] In one aspect, polynucleotides or nucleic acids encoding the antigen
binding
proteins disclosed herein are provided. Methods of making a antigen binding
protein comprising
expressing these polynucleotides or nucleic acids are also provided.
[0319] Polynucleotides encoding the antigen binding proteins disclosed herein
are
typically inserted in an expression vector for introduction into host cells
that may be used to
produce the desired quantity of the antigen binding proteins. Accordingly, in
certain aspects, the
invention provides expression vectors comprising polynucleotides disclosed
herein and host cells
comprising these vectors and polynucleotides.
[0320] The term "vector" or "expression vector" is used herein to mean vectors
used in
accordance with the present invention as a vehicle for introducing into and
expressing a desired
gene in a cell. As known to those skilled in the art, such vectors may readily
be selected from the
group consisting of plasmids, phages, viruses and retroviruses. In general,
vectors compatible
with the instant invention will comprise a selection marker, appropriate
restriction sites to
facilitate cloning of the desired gene and the ability to enter and/or
replicate in eukaryotic or
prokaryotic cells.
[0321] Numerous expression vector systems may be employed for the purposes of
this
invention. For example, one class of vector utilizes DNA elements which are
derived from animal
viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia
virus, baculovirus,
retroviruses (e.g., RSV, MMTV, MOMLV or the like), or SV40 virus. Others
involve the use of
polycistronic systems with internal ribosome binding sites. Additionally,
cells which have
integrated the DNA into their chromosomes may be selected by introducing one
or more markers
which allow selection of transfected host cells. The marker may provide for
prototrophy to an
auxotrophic host, biocide resistance (e g , antibiotics) or resistance to
heavy metals such as
copper. The selectable marker gene can either be directly linked to the DNA
sequences to be
expressed or introduced into the same cell by co-transformation. Additional
elements may also be
needed for optimal synthesis of mRNA. These elements may include signal
sequences, splice
signals, as well as transcriptional promoters, enhancers, and termination
signals. In some
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embodiments, the cloned variable region genes are inserted into an expression
vector along with
the heavy and light chain constant region genes (e.g., human constant region
genes) synthesized
as discussed above.
[0322] In other embodiments, the antigen binding proteins may be expressed
using
polycistronic constructs. In such expression systems, multiple gene products
of interest such as
heavy and light chains of antibodies may be produced from a single
polycistronic construct. These
systems advantageously use an internal ribosome entry site (IRES) to provide
relatively high
levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are
disclosed in U.S.
Pat. No. 6,193,980, which is incorporated by reference herein in its entirety
for all purposes. Those
skilled in the art will appreciate that such expression systems may be used to
effectively produce
the full range of polypeptides disclosed in the instant application.
[0323] More generally, once a vector or DNA sequence encoding an antibody, or
fragment
thereof, has been prepared, the expression vector may be introduced into an
appropriate host cell.
That is, the host cells may be transformed. Introduction of the plasmid into
the host cell can be
accomplished by various techniques well known to those of skill in the art.
These include, but are
not limited to, transfection (including electrophoresis and electroporation),
protoplast fusion,
calcium phosphate precipitation, cell fusion with enveloped DNA,
microinjection, and infection
with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors"
Chapter 24.2, pp.
470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass.
1988). Plasmid
introduction into the host can be by electroporation. The transformed cells
are grown under
conditions appropriate to the production of the light chains and heavy chains,
and assayed for
heavy and/or light chain protein synthesis. Exemplary assay techniques include
enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated
cell sorter
analysis (FACS), immunohistochemistry and the like.
[0324] As used herein, the term "transformation" shall be used in a broad
sense to refer to
the introduction of DNA into a recipient host cell that changes the genotype
and consequently
results in a change in the recipient cell.
[0325] Along those same lines, "host cells" refers to cells that have been
transformed with
vectors constructed using recombinant DNA techniques and encoding at least one
heterologous
gene. In descriptions of processes for isolation of polypeptides from
recombinant hosts, the terms
"cell" and "cell culture" are used interchangeably to denote the source of
antibody unless it is
clearly specified otherwise. In other words, recovery of polypeptide from the
"cells- may mean
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either from spun down whole cells, or from the cell culture containing both
the medium and the
suspended cells.
[0326] In one embodiment, a host cell line used for antibody expression is of
mammalian
origin. Those skilled in the art can determine particular host cell lines
which are best suited for
the desired gene product to be expressed therein. Exemplary host cell lines
include, but are not
limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA
(human
cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with
SV40 T
antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK
(hamster
kidney line), SP2/0 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells),
RAJI (human
lymphocyte), 293 (human kidney) and the like. In one embodiment, the cell line
provides for
altered glycosylation, e.g., afucosylation, of the antibody expressed
therefrom (e.g., PER.C6
(Crucell) or FUT8-knock-out CHO cell lines (Potelligent cells) (Biowa,
Princeton, N.J.)). Host
cell lines are typically available from commercial services, e.g., the
American Tissue Culture
Collection, or from published literature
[0327] In vitro production allows scale-up to give large amounts of the
desired
polypeptides. Techniques for mammalian cell cultivation under tissue culture
conditions are
known in the art and include homogeneous suspension culture, e.g., in an
airlift reactor or in a
continuous stirrer reactor, or immobilized or entrapped cell culture, e.g., in
hollow fibers,
microcapsules, on agarose microbeads or ceramic cartridges. If necessary
and/or desired, the
solutions of polypeptides can be purified by the customary chromatography
methods, for example
gel filtration, ion-exchange chromatography, chromatography over DEAE-
cellulose and/or
(immuno-) affinity chromatography.
[0328] Genes encoding the antigen binding proteins featured in the invention
can also be
expressed in non-mammalian cells such as bacteria or yeast or plant cells. In
this regard it will be
appreciated that various unicellular non-mammalian microorganisms such as
bacteria can also be
transformed, i e , those capable of being grown in cultures or fermentation
Bacteria, which are
susceptible to transformation, include members of the enterobacteriaceae, such
as strains of
Escherichia coil or Salmonella; Bacillaceae, such as Bacillus subtilis;
Pneumococcus;
Streptococcus, and Haemophilus influenzae. It will further be appreciated
that, when expressed
in bacteria, the proteins can become part of inclusion bodies. The proteins
must be isolated,
purified and then assembled into functional molecules.
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[0329] In addition to prokaryotes, eukaryotic microbes may also be used.
Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used among
eukaryotic
microorganisms, although a number of other strains are commonly available. For
expression in
Saccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature,
282:39 (1979);
Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)),
is commonly used.
This plasmid already contains the TRP1 gene which provides a selection marker
for a mutant
strain of yeast lacking the ability to grow in tryptophan, for example ATCC
No. 44076 or PEP4-
1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as a
characteristic of the yeast
host cell genome then provides an effective environment for detecting
transformation by growth
in the absence of tryptophan.
Engineering and Optimization of Antigen Binding Proteins
[0330] The antigen binding proteins of the disclosure may be engineered or
optimized. As
used herein, "optimized" or "optimization" refers to the alteration of an
antigen binding protein
to improve one or more functional properties. Alteration includes, but is not
limited to, deletions,
substitutions, additions, and/or modifications of one or more amino acids
within an antigen
binding protein.
[0331] As used herein, the term "functional property" is a property of an
antigen binding
protein for which an improvement (e.g., relative to a conventional antigen
binding protein, such
as an antibody) is desirable and/or advantageous to one of skill in the art,
e.g., in order to improve
the manufacturing properties or therapeutic efficacy of an antigen binding
protein. In one
embodiment, the functional property is stability (e.g., thermal stability). In
another embodiment,
the functional property is solubility (e.g., under cellular conditions). In
yet another embodiment,
the functional property is aggregation behavior. In still another embodiment,
the functional
property is protein expression (e.g., in a prokaryotic cell). In yet another
embodiment the
functional property is refolding behavior following inclusion body
solubilization in a
manufacturing process. In certain embodiments, the functional property is not
an improvement in
antigen binding affinity. In another embodiment, the improvement of one or
more functional
properties has no substantial effect on the binding affinity of the antigen
binding protein.
[0332] In certain embodiments, the antigen binding protein of the disclosure
is an scFv
and is optimized by identifying preferred amino acid residues to be
substituted, deleted, and/or
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added at amino acid positions of interest (e.g., amino acid positions
identified by comparing a
database of scFv sequences having at least one desirable property, e.g., as
selected with Quality
Control (QC) assay, versus a database of mature antibody sequences, e.g., the
Kabat database) in
an antigen binding protein. Thus, the disclosure further provides
"enrichment/exclusion" methods
for selecting a particular amino acid residue. Still further, the disclosure
provides methods of
engineering antigen binding proteins (e.g., scFvs) by mutating particular
framework amino acid
positions identified using the "functional consensus" approach described
herein. In certain
embodiments, the framework amino acid positions are mutated by substituting
the existing amino
acid residue by a residue which is found to be an "enriched" residue using the
"enrichment/exclusion" analysis methods described herein. In one aspect, the
disclosure provides
a method of identifying an amino acid position for mutation in a single chain
antibody (scFv), the
scFv having VH and VL amino acid sequences, the method comprising: a) entering
the scFv VH,
VL or VH and VL amino acid sequences into a database that comprises a
multiplicity of antibody
VH, VL or VI-1 and VL amino acid sequences such that the scFv VH, VL or VH and
VL amino
acid sequences are aligned with the antibody VH, VL or VH and VL amino acid
sequences of the
database; b) comparing an amino acid position within the scFv VII or VL amino
acid sequence
with a corresponding position within the antibody VH or VL amino acid
sequences of the
database; c) determining whether the amino acid position within the scFv VH or
VL amino acid
sequence is occupied by an amino acid residue that is conserved at the
corresponding position
within the antibody VH or VL amino acid sequences of the database; and d)
identifying the amino
acid position within the scFv VII or VL amino acid sequence as an amino acid
position for
mutation when the amino acid position is occupied by an amino acid residue
that is not conserved
at the corresponding position within the antibody VH or VL amino acid
sequences of the database.
ScFy optimization is described in further detail in W02008110348,
W02009000099,
W02009000098, and W02009155725, all of which are incorporated herein by
reference.
[0333] In those aspects of the disclosure where the presence of an Fc domain
is
practicable, the antigen binding protein may comprise an Fc domain which is
modified such that
it does not induce cytotoxic immune responses and/or does not activate
complement. For example,
one or more substitutions may be introduced into the Fc domain so that its
ADCC/ADCP or CDC
effector function is inactivated. Such antigen binding protein has the
advantage of increased half-
life when compared to antibody fragments with a molecular weight below 60 kDa,
without
mediating mediate cytotoxic immune responses.
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Chemical and/or biological modifications
[0334] In one aspect, the antigen binding protein is chemically and/or
biologically
modified. For example, the antigen binding protein may be glycosylated,
phosphorylated,
hydroxylated, PEGylated, HESylated, PASylated, sulfated, labeled with dyes
and/or
radioisotopes, conjugated with enzymes and/or toxins, and/or Albumin binding
or fusion
technology. Likewise, any nucleic acid sequence, plasmid or vector and/or host
cell described
herein may be modified accordingly.
[0335] Such modification may for example be done to optimize pharmacokinetics,
the
water solubility or to lower side effects. For example, PEGylation,
PASylation, HESylation and/or
the fusion to serum albumin may be applied to slow down renal clearance,
thereby increasing
plasma half-life time of the antigen binding protein. In ne embodiment, the
antigen binding
molecules of the disclosure are operably linked to human serum albumin. In one
embodiment, a
modification adds a different functionality to the antigen binding protein,
for example, a detection
label for diagnostics or a toxin to combat cancer cells even more efficiently.
[0336] In one embodiment, the antigen binding protein is glycosylated.
Glycosylation
refers to a process that attaches carbohydrates to proteins. In biological
systems, this process is
performed enzymatically within the cell as a form of co-translational and/or
post- translational
modification. A protein can also be chemically glycosylated. The carbohydrates
may be N-linked
to a nitrogen of asparagine or arginine side-chains; 0-linked to the hydroxy
oxygen of serine,
threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains; employ
xylose, fucose,
mannose, and N-acetylglucosamine attached to a phospho-serine; and/or adding
mannose sugar
to a tryptophan residue found in a specific recognition sequence.
Glycosylation patterns may, e.g.,
be controlled by choosing appropriate cell lines, culturing media, protein
engineering
manufacturing modes and process strategies (see., HOS SLER, P. Optimal and
consistent protein
glycosylation in mammalian cell culture. Glycobiology 2009, vol. 19, no. 9, p.
936-949.). In some
embodiments, the glycosylation patterns of the antigen binding proteins
described herein are
modified to enhance ADCC and CDC effector function.
[0337] The antigen binding protein may be engineered to control or alter the
glycosylation
pattern, e.g., by deleting and/or adding of one or more glycosylation sites.
The creation of
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glycosylation sites can e.g., be accomplished by introducing the corresponding
enzymatic
recognition sequence into the amino acid sequence of the antigen binding
protein.
[0338] In some embodiments, the antigen binding protein is PEGylated.
PEGylation may
alter the pharmacodynamic and pharmacokinetic properties of a protein.
Additionally, PEGylation
may reduce the immunogenicity by shielding the PEGylated antigen binding
protein from the
immune system and/or alter its pharmacokinetics by, e.g., increasing the in
vivo stability of the
antigen binding protein, protecting it from proteolytic degradation, extending
its half-life time and
by altering its biodistribution. Typically, polyethylene-glycol (PEG) of an
appropriate molecular
weight is covalently attached to the protein. Similar effects may be achieved
using PEG mimetics,
e.g., HESylating, PASylating, or XTENylating the antigen binding protein.
HESylation utilizes
hydroxyethyl starch ("HES") derivatives. During PASylation, the antigen
binding protein is linked
to conformationally disordered polypeptide sequences composed of the amino
acids proline (P),
alanine (A) and serine (S), and XTENylation employs a similar, intrinsically
disordered XTEN-
polypepti de
[0339] In certain embodiments, the antigen binding protein is labelled with or
conjugated
to a second moiety which attributes one or more ancillary functions to the
antigen binding protein.
For example, the second moiety may have an additional immunological effector
function, be
effective in drug targeting or useful for detection. The second moiety can,
e.g., be chemically
linked or fused genetically to the antigen binding protein using known methods
in the art. As used
herein, the term ''label" refers to any substance or ion which is indicative
of the presence of the
antigen binding protein when detected or measured by physical or chemical
means, either directly
or indirectly. For example, the label may be directly detectable by, without
being limited to, light
absorbance, fluorescence, reflectivity, light scatter, phosphorescence, or
luminescence properties,
molecules or ions detectable by their radioactive properties or molecules or
ions detectable by
their nuclear magnetic resonance or paramagnetic properties. Examples of
indirect detection
include light absorbance or fluorescence; for example, various enzymes which
cause appropriate
substrates to convert, e.g., from non-light absorbing to light absorbing
molecules, or from non-
fluorescent to fluorescent molecules. A labelled antigen binding protein is
particularly useful for
in vitro and in vivo detection or diagnostic purposes. For example, an antigen
binding protein
labelled with a suitable radioisotope, enzyme, fluorophore or chromophore can
be detected by
radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow
cytometry-
based single cell analysis (e.g., FACS analysis), respectively. Similarly, the
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vectors disclosed herein can be labeled for detection or diagnostic purposes,
e.g., using labelled
fragments thereof as probes in hybridization assays.
[0340] Non-limiting examples of second moieties include radioisotopes (35S,
32P, 14C,
18F, and/or 1251), apoenzymes, enzymes (e.g., alkaline phosphatase,
horseradish peroxidase,
beta-galactosidase and/or angiogenin), co-factors, peptide moieties (e.g., a
HI S -t ag), proteins (e.g.
lectin, serum albumin), carbohydrates (e.g., mannose-6-phosphate tags),
fluorophores (e.g.,
fluorescein isothiocyanate (FITC)), phycoerythrin, green/blue/red or other
fluorescent proteins,
allophycocyanin (APC), chromophores, vitamins (e.g., biotin), chelators,
antimetabolites (e.g.,
methotrexate), toxins (e.g. a cytotoxic drug, or a radiotoxin).
[0341] In one aspect, the invention relates to drug conjugates (in particular
antibody-drug
conjugates ADCs) comprising the antigen binding proteins described herein
conjugated to a toxin
which further enhances efficient killing of specific cells, such as e.g., MAGE-
A4 positive cells.
The toxin moiety is typically a small molecular weight moiety, such as
M1VIAEAVIMAF, DM1,
chaliceamicin, anthracycline toxins, taxol, gramicidin D and/or colchicine,
which may be linked
via a peptide linker to the antigen binding protein.
[0342] The toxin may be conjugated non-site-specifically or site-specifically
to the
antigen binding protein. Non-site-specific conjugation typically involves the
use of chemical
linkers, e.g., with maleimide functionality, that mediate conjugation to
lysine or cysteine amino
acid side chains of the antibody. Site- specific conjugation may be achieved
using chemical,
chemo-enzymatic, or enzymatic conjugations known in the art, e.g., employing
bifunctional
linkers, bacterial transglutaminase or sortase enzymes, linkers allowing
Pictet-Spengler chemistry
on formyl-glycine forming enzyme modified antigen binding proteins, or glycan-
remodeled
antigen binding proteins.
Methods of Administering Antigen Binding Proteins
[0343] Methods of preparing and administering antigen binding proteins of the
disclosure
as well as the nucleic acids described herein, the vectors described herein,
the host cell cells
described herein or the compositions described herein to a subject are well
known to or are readily
determined by those skilled in the art. The route of administration of the
antigen binding proteins
of the current disclosure may e.g., be oral, parenteral, by inhalation, or
topical. The term parenteral
as used herein includes intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous,
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rectal or vaginal administration. The term intraocular as used herein
includes, but is not limited
to, subconjunctival, intravitreal, retrobulbar, or intracameral. The term
topical as used herein
includes, but is not limited to, administration with liquid or solution eye
drops, emulsions (e.g.,
oil-in-water emulsions), suspensions, and ointments.
[0344] While all these forms of administration are clearly contemplated as
being within
the scope of the current disclosure, a form for administration would be a
solution for injection.
Usually, a suitable pharmaceutical composition for injection may comprise a
buffer (e.g., acetate,
phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a
stabilizer agent (e.g.,
human albumin), etc. However, in other methods compatible with the teachings
herein, the
modified antibodies can be delivered directly to the site of the adverse
cellular population thereby
increasing the exposure of the diseased tissue to the therapeutic agent.
[0345] Effective doses of the compositions of the present disclosure, for the
treatment of
the related conditions vary depending upon many different factors, including
means of
administration, target site, physiological state of the patient, whether the
patient is human or an
animal, other medications administered, and whether treatment is prophylactic
or therapeutic.
Usually, the patient is a human, but non-human mammals, including transgenic
mammals, can
also be treated. Treatment dosages may be titrated using routine methods known
to those of skill
in the art to optimize safety and efficacy.
[0346] As previously discussed, the antigen binding proteins of the present
disclosure,
conjugates or recombinants thereof may be administered in a pharmaceutically
effective amount
for the in vivo treatment of mammalian disorders. In this regard, it will be
appreciated that the
disclosed antigen binding proteins will be formulated to facilitate
administration and promote
stability of the active agent.
[0347] Pharmaceutical compositions in accordance with the present disclosure
typically
include a pharmaceutically acceptable, non-toxic, sterile carrier such as
physiological saline,
nontoxic buffers, preservatives and the like. For the purposes of the instant
application, a
pharmaceutically effective amount of the antigen binding proteins shall be
held to mean an
amount sufficient to achieve effective binding to an antigen and to achieve a
benefit, e.g., to
ameliorate symptoms of a disease or disorder or to detect a substance or a
cell. In the case of
tumor cells, the antigen binding proteins will typically be capable of
interacting with selected
immunoreactive antigens on neoplastic or immunoreactive cells and provide for
an increase in the
death of those cells. Of course, the pharmaceutical compositions of the
present disclosure may be
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administered in single or multiple doses to provide for a pharmaceutically
effective amount of the
modified binding polypeptide.
[0348] In keeping with the scope of the present disclosure, the antigen
binding proteins of
the disclosure may be administered to a human or other animal in accordance
with the
aforementioned methods of treatment in an amount sufficient to produce a
therapeutic or
prophylactic effect. The antigen binding proteins of the disclosure can be
administered to such
human or other animal in a conventional dosage form prepared by combining the
antigen binding
proteins of the disclosure with a conventional pharmaceutically acceptable
carrier or diluent
according to known techniques. It will be recognized by one of skill in the
art that the form and
character of the pharmaceutically acceptable carrier or diluent is dictated by
the amount of active
ingredient with which it is to be combined, the route of administration and
other well-known
variables. Those skilled in the art will further appreciate that a cocktail
comprising one or more
species of antigen binding proteins described in the current disclosure may
prove to be particularly
effective Similarly, the nucleic acids described herein, the vectors described
herein, the host cell
cells described herein (in particular the immune cells bearing a CAR) or the
compositions
described herein may be administered to a human or other animal in accordance
with the methods
of treatment described above in an amount sufficient to produce a therapeutic
or prophylactic
effect.
[0349] "Efficacy" or "in vivo efficacy" as used herein refers to the response
to a therapy
by the pharmaceutical composition of the disclosure, using e.g., standardized
response criteria,
such as standard ophthalmological response criteria. The success or in vivo
efficacy of the therapy
using a pharmaceutical composition of the disclosure refers to the
effectiveness of the composition
for its intended purpose, i.e., the ability of the composition to cause its
desired effect. The in vivo
efficacy may be monitored by established standard methods for the specific
diseases. In addition,
various disease specific clinical chemistry parameters and other established
standard methods
may be used.
[0350] In some embodiments, the compounds and cells described herein are
administered
in combination with one or more different pharmaceutical compounds. Generally,
therapeutic use
of the compounds and cells described herein may be in combination with one or
more therapies
selected from the group of antibody therapy, chemotherapy, cytokine therapy,
dendritic cell
therapy, gene therapy, hormone therapy, laser light therapy, radiation therapy
or vaccine therapy.
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Methods of Treating Cancer or Viral Infections
103511 Provided herein are methods of treating cancer or viral infections with
the antigen
binding proteins of the disclosure (e.g., an antigen binding protein
comprising a Fab domain which
binds a cell surface protein of an immune cell linked to a first and second
pMEC binding domain).
In certain embodiments, the cancer is caused by a viral infection.
[0352] In certain embodiments of the antigen binding protein of the
disclosure, the target
pMEC binding domain specifically targets an MEC restricted peptide derived of
a tumor antigen
or a viral antigen.
[0353] In one aspect, the disclosure provides a method for killing a target
cell comprising
a major histocompatibility complex (MEC) presenting a neoantigen, the method
comprising: a)
contacting a plurality of cells comprising immune cells and the target cell
with the antigen binding
protein described above, wherein said antigen binding protein specifically
binds to the pMEC on
the surface of the target cell and to CD3 on the surface of the immune cells;
b) forming a specific
binding complex through the antigen binding protein interactions with the
target cells and the
immune cells, thereby activating the immune cells; and c) killing the target
cell with the activated
immune cells.
[0354] In one aspect, the disclosure provides a method of treating cancer
comprising the
step of administering the antigen binding protein described above to a patient
in need thereof
Kits
[0355] Also contemplated are kits comprising at least one nucleic acid library
or antigen
binding protein as described herein, typically together with a packaged
combination of reagents
with instructions. In one embodiment, the kit includes a composition
containing an effective
amount of said antigen binding protein in unit dosage form. Such kit may
comprise a sterile
container comprising the composition; non-limiting examples of such containers
include, without
being limited to, vials, ampoules, bottles, tubes, syringes, blister-packs. In
some embodiments,
the composition is a pharmaceutical composition and the containers is made of
a material suitable
for holding medicaments. In one embodiment, the kit may comprise in a first
container the antigen
binding protein in lyophilized form and a second container with a diluent
(e.g., sterile water) for
reconstitution or dilution of the antigen binding protein. In some
embodiments, said diluent is a
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pharmaceutically acceptable diluent. In one embodiment, the kit is for
diagnostic purposes and
the antigen binding protein is formulated for diagnostic applications. In one
embodiment, the kit
is for therapeutic purposes and the antigen binding protein is formulated for
therapeutic
applications.
[0356] Typically, the kit will further comprise a separate sheet, pamphlet or
card supplied
in or with the container with instructions for use. If the kit is intended for
pharmaceutical use, it
may further comprise one or more of the following: information for
administering the composition
to a subject having a related disease or disorder and a dosage schedule,
description of the
therapeutic agent, precautions, warnings, indications, counter-indications,
overdosage
information and/or adverse reactions.
[0357] It will be readily apparent to those skilled in the art that other
suitable
modifications and adaptations of the methods described herein may be made
using suitable
equivalents without departing from the scope of the embodiments disclosed
herein. Having now
described certain embodiments in detail, the same will be more clearly
understood by reference
to the following examples, which are included for purposes of illustration
only and are not
intended to be limiting.
[0358] Table 4 ¨ Antigen binding protein amino acid sequences. CDR sequences
are
highlighted in bold underlined text.
SEQ ID NO. Compound Sequence
> CDR-1 HC
EVQLVESGGGSVQPGGSLRL SCAASG
FTFSTYAMNWVRQAPGKGLEWVGRIRSKA
NNYATYYADSVKGRFTISRDD SKNTLYL QM
SEQ ID NO.: 40 NSLRAEDTATYYCVRHGNFGDSYVSWFAY
CDR- 1
WGQGTTVTVS SAS TKGP SVFPLAPS SK S T SG
GTAALGCLVKDYFPEPVTVSWN S GALT SGV
HTFPAVLQ S SGLYSLS SVVTVP S S SLGTQTYI
CNVNHKP SNTKVDKRVEPK S C
SEQ ID NO.: 41 > CDR-I LC
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EVQLVES GGGLAQAGGSLRVSC VAS GRPFT
KYAWGWFRQAPGKAREFVATITWDGGKT
DYAD SVKGRF TI SKD SAENSIYL QMNSLKPE
DTAVYYCAADRNYCVGHRCYVRPDDYDY
WGQGTQVTVSSGGGGSAVVTQEPSLTVSPG
GT VTLTCGSSTGA VTTSN YAN WVQQKPGK
SPRGLIGGTNKRAPGVPARFSGSLLGGKAA
LTISGAQPEDEADYYCALWYSNHWVFGGG
TKLTVLGTVAAP S VFIFPP SDEQLK S GTA S V
VCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
> CDR-2 HC
EVQLVES GGGS VQP GGSLRL SCAA SGF TF ST
YAMNWVRQAPGKGLEWVGRIRSKANNYA
SEQ ID NO 42 TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDT A TYYCVRHGNFGD SYVSWFAYWGQ
GTTVTVSS A STKGPSVFPT,APSSKSTSGGTA
AL GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKP SNTKVDKRVEPKS C
CDR-2 > CDR-2 LC
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTS
NYANWVQQKPGKSPRGLIGGTNKRAPGVP
ARFSGSLLGGKAALTISGAQPEDEADYYCA
SEQ ID NO 43 LW YSN HW VFCiGGTKLT VLGT VAAP S
VFIFP
PSDEQLK SGTA SVVCLLNNFYPREAK VQWK
VDNALQSGNSQESVTEQD SKDSTYSLS STLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFN
RGECGGGGSEVQLVESGGGLAQAGGSLRVS
CVASGRPF TKYAW GWFRQAP GKAREF VAT
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ITWDGGKTDYADSVKGRFTISKDSAENSIY
LQMNSLKPEDTAVYYCAADRNYCVGHRC
YVRPDDYDYWGQGTQVTVS S
> CDR-3 HC
EVQLVESGGGSVQPGGSLRLSCAASGF TF ST
YAIVINWVRQAPGKGLEWVGRIRSKANNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTATYYCVRHGNFGDSYVSWFAYWGQ
GTTVTVSSASTKGPSVFPLAPSSKSTSGGTA
SEQ ID NO.: 44 ALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCGGGGSEVQLVES
GGGLAQAGGSLRVSCVASGRPFTKYAWGW
FRQAPGKAREFVATITWDGGKTDYADSVK
CDR-3 GRFTISKDSAENSIYLQMN SLKPEDTAVYYC
AADRNYCVGHRCYVRPDDYDYWGQGTQ
VTVSS
> CDR-3 LC
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTS
NYANWVQQKPGKSPRGLIGGTNKRAPGVP
ARFSGSLLGGKAALTISGAQPEDEADYYCA
SEQ ID NO.: 45 LWYSNHWVFGGGTKLTVLGTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQD SKDSTYSLS STLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFN
RCiEC
> CDR-4 HC
SEQ ID NO.: 46 EVQLVESGGGLAQAGGSLRVSCVASGRPFT
CDR-4 KYAWGWFRQAPGKAREFVATITWDGGKT
DYADSVKGRFTISKDSAENSIYLQMNSLKPE
DTAVYYCAADRNYCVGHRCYVRPDDYDY
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WGQGTQVT VS S GGGGSEVQLVES GGGS VQ
PGGSLRLSCAASGFTFSTYAMNWVRQAPGK
GLEWVGRIRSKANNYATYYADSVKGRFTIS
RDDSKNTLYLQMNSLRAEDTATYYCVRHG
NFGDSYVSWFAYWGQGTTVTVSSASTKGP
S VFPL AP S SKST SG G TAAL GCL VKD YFPEP V
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSS SLGTQTYICNVNHKPSNTKVDKRVE
PKSC
> CDR-4 LC
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTS
NYANWVQQKPGKSPRGLIGGTNKRAPGVP
ARFSGSLLGGKAALTISGAQPEDEADYYCA
SEQ ID NO.: 47 LWYSNHWVFGGGTKLTVLGTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQD SKDSTYSLS STLT
LSKADYEKHKVYACEVTHQGLSSPVTK SFN
RGFC
> CDR-5 HC
EVQLVESGGGSVQPGGSLRLSCAASGFTFST
YA1VINWVRQAPGKGLEWVGRIRSKANNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTATYYCVRHGNFGDSYVSWFAYWGQ
SEQ ID NO. : 48
CDR-5
GTTVTVSSASTKGPSVFPLAPSSKSTSGGTA
AL GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NITKPSNTKVDKRVEPK SCGGGG SEVQLVES
GGGLAQAGGSLRVSCVASGRPFTKYAWGW
FRQAPGKAREFVATITWDGGKTDYADSVK
GRFTISKDSAENSIYLQMNSLKPEDTAVYYC
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AADRNYCVGHRCYVRPDDYDYWGQGTQ
VTVSS>
> CDR-5 LC
EVQLVESGGGLAQAGGSLRV SC VASGRPFT
KYAWGWFRQAPGKAREFVATITWDGGKT
DYADSVKGRFTISKDSAENSIYLQMNSLKPE
DTAVYYCAADRNYCVGHRCYVRPDDYDY
WGQGTQVTVSSGGGGSAVVTQEPSLTVSPG
SEQ ID NO.: 49 GTVTLTCGSSTGAVTTSNYANWVQQKPGK
SPRGLIGGTNKRAPGVPARFSGSLLGGKAA
LTISGAQPEDEADYYCALWYSNHWVFGGG
TKLTVLGTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
> CDR-6 HC
EVQLVESGGGSVQPGGSLRLSCAASGFTFST
YA1VINWVRQAPGKGLEWVGRIRSKANNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTATYYCVRHGNFGDSYVSWFAYWGQ
GT TVTVS S AS TKGP SVFPLAP S SKSTSGGTA
SEQ ID NO.: 50 ALGCLVKDYFPEPVTVSWNSGALTSGVHTF
CDR-6 PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCGGGGSEVQLVES
GGGLAQAGGSLRVSCVASGRPFTKYAWGW
FRQAPCiKAREF VATITWDGGKTDYADSVK
GRFTISKDSAENSIYLQMNSLKPEDTAVYYC
AADRNYCVGHRCYVRPDDYDYWGQGTQ
VTVSS>
SEQ ID NO.: 51 > CDR-6 LC
79
CA 03240046 2024- 6- 4
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PCT/EP2022/085689
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTS
NYANWVQQKPGKSPRGLIGGTNKRAPGVP
ARF SGSLLGGKAALTISGAQPEDEADYYCA
LWY SNHWVF GGGTKLTVL GTVAAP S VF IF'P
PSDEQLKSGTAS V VCLLNNF YPREAK VQWK
VDNALQSGN SQES VTEQD SKDSTY SLS SILT
L SKADYEKHKVYACEVTHQGLS SPVTKSFN
RGECGGGGSEVQLVESGGGLAQAGGSLRVS
CVASGRPF TKYAW GWFRQAP GKAREF VAT
ITWDGGKTDYADSVKGRF TISKDSAENSIY
LQMNSLKPEDTAVYYCAADRNYCVGHRC
YVRPDDYDYWGQGTQVTVS S
> CDR-7 HC
FVQI NES GGGI ,VQPGGSI ,RI SC A A SGFTFST
YAMNWVRQAPGKGLEWVGRIRSKYNNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVS S AS TKGP SVFPLAP S SKSTSGGTA
AL GCLVKDYFPEPVTVSWNSGALT SGVHTF
SEQ ID NO .52 PAVLQSSGLYSLS SVVTVPS S SLGTQTYICNV
NHKP SNTKVDKRVEPK S CGGGGS SYELTQP
PSVSVSPGQTASITCTADTLSRSYASWYQQK
PGQSPVLVIYRDTSRPSGIPERF SG SNSGNT A
TLTIS GT QAMDEADYYCATRP S S GSNFQLF
CDR-7 GGGTKLTVLGGGGGGSGGGGSGGGGSGGG
GSESQVLESGGG SVQPGG SLRL SC TVSGF SL
SNYAMSWVRQ AP GKGLEYIGIV S S GGT TYY
A SW AKGRF TISKDTSKNTVYLQMNSLRAED
TA SYYCAKDLYYGPT TY SAFNLWGQ GT S V
TVS S
> CDR-7 LC
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGK SPRGLIGGTNKRAPG
VPARF SGSLLGGKAALTISGAQPEDEADYYC
SEQ ID NO. 53 ALWYSNHWVFGGGTKLTVLGQPKAAP S VT
LFPP S SEELQANKATLVCLISDFYPGAVTVA
WKADS SPVKAGVETTTP SKQ SNNKYAAS SY
L SLTPEQWK SHR SYSCQVTHEG STVEK TVA
PTEC S
CA 03240046 2024- 6- 4
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> CDR-8 HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFST
YA1VINWVRQAPGKGLEWVGRIRSKYNNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVS S AS TKGP SVFPLAP S SKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLS SVVTVPS S SLGTQTYICNV
SEQ ID NO.:54 NHKP SNTKVDKRVEPKSCGGGGSSYELTQP
PSVSVSPGQTASITCTADTLSRSYASWYQQK
PGQ SP VLVIYRDTSRPSGIPERF SGSN SGN TA
TLTISGTQAMDEADYYCATRPSSGSNFOLF
GGGTKLTVLGGGGGGSGGGGSGGGGSGGG
GSES QVLESGGGSVQPGGSLRL SC TVSGF SL
SNYAMSWVRQAPGKGLEYIGIVSSGGTTYY
CDR-8 ASWAKGRFTISKDTSKNTVYLQMNSLRAED
TASYYCAKDLYYGPTTYSAFNLWGQGTSV
TVSS
> CDR-8 LC
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGKSPRGLIGGTNKRAPG
VPARFSGSLLGGKAALTISGAQPEDEADYYC
ALWYSNHWVFGGGTKLTVLGQPKAAPSVT
LFPP S SEELQANKATLVCLISDFYPGAVTVA
SEQ ID NO..55 WKADS SP VKAGVETTTP SKQ SNNKYAAS S
Y
L SLTPEQWKSHRSYSCQVTHEGSTVEKTVA
PTECSGGGGS SYELTQPP SVSVSPGQTASITC
TADTLSRSYASWYQQKPGQSPVLVIYRDTS
RP S GIPERF SGSNSGNTATLTISGTQAMDEA
DYYCATRPSSGSNFOLFGGGTKLTVLGGGG
GGSGGGGSGGGGSGGGGSESQVLESGGGSV
QPGGSLRLSCTVSGFSLSNYAMSWVRQAPG
81
CA 03240046 2024- 6- 4
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PCT/EP2022/085689
KGLEYIGIV SS GGTTYYA SWAKGRF TISKD
TSKNTVYLQMNSLRAEDTASYYCAKDLYY
GPTTYSAFNLWGQ GT SVTVS S
> CDR-9 HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFST
YA1VINWVRQAPGKGLEWVGRIRSKANNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
SEQ ID N O. : 56 ALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKP SNTKVDKRVEPK SC GGGGSEVQLVES
GGGLVQPGGSLRLSCVASGRPFTKYAWGW
FRQAP GKAREF VATITW D GGKTDYAD SVK
GRFTISKDSAKN SI YLQMN SLRAEDTAV Y YC
AADRNYCVGHRCYVRPDDYDYWGQGTLV
CDR-9 TVS S
> CDR-9 LC
QAVVTQEP SL TV SPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGK SPRGLIGGTNKRAPG
VPARF SGSLLGGKAALTISGAQPEDEADYYC
ALWY SNHWVF GGGTKLTVLGTVAAP SVFIF
PP SDEQLK SGTASVVCLLNNFYPREAKVQW
SEQ ID NO.: 57 KVDNALQSGNSQESVTEQDSKD STYSLSSTL
TLSKADYEKHKVYACEVTHQGLS SPVTK SF
NRGECGGCJCISEVQLVESGGCiL V QPGGSLRL
SCVASGRPFTKYAWGWFRQAPGKAREFVA
TITWD GGKTD YAD SVKGRF TISKD SAKNSI
YLQMNSLRAEDTAVYYCAADRNYCVGHR
CYVRPDDYDYWGQ GTLVT V S
SEQ ID NO 58 CDR-10 > CDR-10 HC
82
CA 03240046 2024- 6- 4
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PCT/EP2022/085689
EVQLVESGGGLVQP GGSLRL SC AASGF TF S T
YA1VINWVRQAPGKGLEWVGRIRSKYNNYA
TYVADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVS S AS TKGP S VFPLAP S SKSTSGGTA
ALG CLVKD YFPEP V TV S W N SGALTSGVHTF
PAVLQSSGLYSLS SVVTVPS S SLGTQTYICNV
NHKPSNTKVDKRVEPKSCGGGGSEVQLVES
GGGLVQPGGSLRLSCVASGRPFTKYAWGW
FRQAPGKAREFVATITWDGGKTDYADSVK
GRFTISKDSAKNSIYLQMNSLRAEDTAVYYC
AADRNYCVGHRCYVRPDDYDYWGQGTLV
TVS S
> CDR-10 LC
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGKSPRGLIGGTNKRAPG
VPARF SG SLLGGK A ALTISG A QPEDEADYYC
A I,WYSNIIWVF GGGTKT ,TVI ,GTV A APSVFTF
PP SDEQLK S GTA S VVCLLNNF YPREAKVQW
SEQ ID NO.: 59 KVDNALQSGNSQESVTEQD SKD STYSLS STL
TLSKADYEKHKVYACEVTHQGLS SPVTK SF
NRGECGGGGSEVQLVESGGGLVQPGGSLRL
S CVA S GRPF TKYAW GWFRQAP GKAREF VA
TITWDGGKTDYAD SVKGRF TISKD SAKN SI
YLQMNSLRAEDTAVYYCAADRNYCVGHR
CYVRPDDYDYWGQGTLVTVS S
> CDR-11 HC
SEQ ID NO.: 60
EVQLVESGGGLVQP GGSLRL SC AASGF TF S T
CDR-11
YA1VINWVRQAPGKGLEWVGRIRSKYNNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGNSYVSWFAYWGQ
83
CA 03240046 2024- 6- 4
WO 2023/110918
PCT/EP2022/085689
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCGGGGSEVQLVES
GGGLVQPGGSLRLSCVASGRPFTKYAWGW
FRQAPGKAREF VATITWDGGKTDYADSVK
GRFTISKDSAKNSIYLQMNSLRAEDTAVYYC
AADRNYCVGHRCYVRPDDYDYWGQGTLV
TV S S
> CDR-11 LC
QAVVTQEPSLTV SPGGTVTLTCRSSTGAVT
TSNYANWVQQKPGQAPRGLIGGTNKRAPG
VPARF SG SLLGGKAAL TISGAQPEDEADYYC
ALWY SNHWVF GGGTKLTVLGTVAAPSVFIF
PP SDEQLK SGTAS VVCLLNNF YPREAKVQW
SEQ ID NO 61 KVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSK ADYEKHKVYACEVTHQGLS SPVTK SF
NR GEC GGGGSF,VQT ,VF,SGGGT ,VQP GGST ,RI ,
SCVAS GRPF TKYAW GWFRQAP GKAREF VA
TITWDGGKTD YAD SVKGRF TISKD SAKNSI
YLQMNSLRAEDTAVYYCAADRNYCVGHR
CYVRPDDYDYWGQGTLVTVS S
> CDR-12 HC
EVQLVESGGGLVQPGGSLRL SC AASGFTF S T
YA1VINWVRQAPGKGLEWVGRIRSKANNYA
SEQ ID NO 62
TY YAD S VKGRF TI SRDD SKN TL YLQMN SLR
CDR- 12
AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVS S AS TKGP SVFPLAP S SKSTSGGTA
AL GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCGGGGSSYELTQP
84
CA 03240046 2024- 6- 4
WO 2023/110918
PCT/EP2022/085689
PSVSVSPGQTASITCTADTLSRSYASWYQQK
PGQSPVLVIYRDTSRPSGIPERFSGSNSGNTA
TLTISGTQAMDEADYYCATSDGSGSNFOLF
GGGTKLTVLGGGGGGSGGGGSGGGGSGGG
GSESQVLESGGGSVQPGGSLRLSCTVSGFSL
SNYAMSWVRQAPGKGLEYIG1VSSGGTTYY
ASWAKGRFTISKDTSKNTVYLQMNSLRAED
TASYYCAKDLYYGPTTYSAFNLWGQGTSV
TVS S
> CDR-12 LC
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGKSPRGLIGGTNKRAPG
VPARFSGSLLGGKAALTISGAQPEDEADYYC
ALWYSNHWVEGGGTKLTVLGTVAAPSVFIF
PP SDEQLK SGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSF
SEQ ID NO 63 NRGECGGGGS SYELTQPP SVSVSPGQTA SIT
CTADTLSRSYASWYQQKPGQSPVLVIYRDT
SRPSGIPERFSGSNSGNTATLTISGTQAMDEA
DYYCATSDGSGSNFOLFGGGTKETVLGGG
GGGSGGGGSGGGGSGGGGSESQVLESGGGS
VQPGGSLRLSCTVSGFSLSNYAMSWVRQAP
GKGLEYIG1VSSGGTTYYASWAKGRFTISK
DT SKNTVYLQMNSLRAEDTASYYCAKDLY
YGPTTYSAFNLWGQGTSVTVSS
> CDR-13 HC
SEQ ID NO 64 CDR-13
EVQLVESGGGLVQPGGSLRLSCAASGFTFST
YAMNWVRQAPGKGLEWVGRIRSKYNNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
CA 03240046 2024- 6- 4
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AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
AL GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCGGGGSSYELTQP
PS VS VSPGQTASITCTADTLSRSYASWYQQK
PGQSPVLVIYRDTSRPSGIPERF SGSNSGNTA
TLTISGTQAMDEADYYCATSDGSGSNFQLF
GGGTKLTVLGGGGGGSGGGGSGGGGSGGG
GSESQVLESGGGSVQPGGSLRLSCTVSGF SL
SNYAMSWVRQAPGKGLEYIGIVSSGGT TYY
ASWAKGRFTISKDTSKNTVYLQMNSLRAED
TASYYCAKDLYYGPTTYSAFNLWGQGTSV
TVSS
> CDR-13 LC
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGK SPRGLIGGTNKRAPG
VP ARF SGSTI,GGK A AT ,TISGA QPFIDEADYYC
ALWYSNHWVFGGGTKLTVLGTVAAPSVFIF
PP SDEQLK S GTASVVCLLNNF YPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLS SPVTK SF
SEQ ID NO.: 65 NRGECGGGGSSYELTQPPSVSVSPGQTASIT
CTADTLSRSYASWYQQKPGQ SP VLVIYRD T
SRPSGIPERF SGSNSGNTATLTISGTQAMDEA
DYYCATSDGSGSNFQLFGGGTKLTVLGGG
GGGSGGGGSGGGGSGGGGSESQVLESGGGS
VQPGGSLRLSCTVSGFSLSNYAMSWVRQAP
GKGLEYIGIVSSGGTTYYASWAKGRF TISK
DT SKNTVYLQMNSLRAEDTASYYCAKDLY
YGPTTYSAFNLWGQGTSVTVSS
86
CA 03240046 2024- 6- 4
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> CDR-14 HC
EVQLVESGGGLVQPGGSLRL SC AASGFTF S T
YA1VINWVRQAPGKGLEWVGRIRSKYNNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGNSYVSWFAYWGQ
GTLVTVS S AS TKGP SVFPLAP S SKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLS SVVTVPS S SLGTQTYICNV
SEQ ID NO.: 66 NHKPSNTKVDKRVEPKSCGGGGSSYELTQP
PSVSVSPGQTASITCTADTLSRSYASWYQQK
PGQ SP VL VIYRDTSRPSGIPERF SGSN S GN TA
TLTISGTQAMDEADYYCATSDGSGSNFOLF
GGGTKLTVLGGGGGGSGGGGSGGGGSGGG
GSESQVLESGGGSVQPGGSLRLSCTVSGFSL
SNYAMSWVRQAPGKGLEYIGIVSSGGTTYY
ASWAKGRFTISKDTSKNTVYLQMNSLRAED
CDR-14
TASYYCAKDLYYGPTTYSAFNLWGQGTSV
TVS S
> CDR-14 LC
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVT
TSNYANWVQQKPGQAPRGLIGGTNKRAPG
VPARFSGSLLGGKAALTISGAQPEDEADYYC
ALWYSNHWVFGGGTKLTVLGTVAAPSVFIF
PP SDEQLK SGTASVVCLLNNFYPREAKVQW
SEQ ID NO.. 67 KVDNALQ SGN SQES V TEQD SKD STY SL S STL
TLSKADYEKHKVYACEVTHQGLS SPVTK SF
NRGECGGGGSSYELTQPPSVSVSPGQTASIT
CTADTLSRSYASWYQQKPGQSPVLVIYRDT
SRPSGIPERFSGSNSGNTATLTISGTQAMDEA
DYYCATSDGSGSNFQLFGGGTKLTVLGGG
GGGSGGGGSGGGGSGGGGSESQVLESGGGS
VQPGGSLRLSCTVSGFSLSNYAMSWVRQAP
87
CA 03240046 2024- 6- 4
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GKGLEYIGIV SSGGTTYYA SWAKGRF TISK
DT SKNTVYL QMNSLRAED TA S YYC AKDLY
YGPT TY SAFNLWGQ GT SVTVSS
> CDR-15 HC
EVQLVESGGGLVQPGGSLRL S C AA SGF TF ST
YA1VINWVRQAPGKGLEWVGRIRSKYNNYA
TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTAVYYCVRHGNFGDSYVSWFAYWGQ
GTLVTVS S AS TKGP SVFPLAP S SKSTSGGTA
AL GCLVKDYFPEPVTV SWNSGALT SGVHTF
PAVLQSSGLY SLSSVVTVPSSSLGTQTYICNV
SEQ ID NO 68 NHKP SNTKVDKRVEPKSCGGGGSSYELTQP
PSVSVSPGQTASITCTADTLSRSYASWYQQK
PGQSPVLVIYRDTSRPSGIPERF SGSNSGNTA
TLTISGTQAMDEADY YCATSDGSGSNFQLF
GGGTKLTVLGGGGGGSGGGGSGGGGSGGG
GSESQVLESGGG SVQPGG SLRL SC TVSGF SL
CDR-15
SNY A MSWVRQ A P GK GT ,EYTGIVSSGGTTYY
ASWAKGRF TISKDTSKNTVYLQMNSLRAED
TASYYCAKDLYYGPTTQSAFNLWGQGTSV
TVSS
> CDR-15 LC
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT
TSNYANWVQQKPGKSPRGLIGGTNKRAPG
VPARF SGSLLGGKAALTISGAQPEDEADYYC
SEQ ID NO 69 ALWYSNHW VFGGGTKLTVLGQPKAAP S VT
LFPP S SEELQANK ATLVCLISDFYPGAVTVA
WKADS SPVKAGVETTTP SKQ SNNKYAAS SY
L SLTPEQWKSHRSYSCQVTHEGSTVEKTVA
PTEC SGGGGS SYELTQPP SVSVSPGQTASITC
TADTLSRSYA SWYQ QKP G Q SPVLVIYRD TS
88
CA 03240046 2024- 6- 4
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RP S GIPERF SGSNSGNTATLTISGTQAMDEA
DYYCAT SD GS GSNFQLF GGGTKLTVLGGG
GGGS GGGGS GGGGS GGGG SE S QVLE S GGGS
VQPGGSLRLSCTVSGF SLSNYAMSWVRQAP
GKGLEYIG1V SSGGTTYYASWAKGRF TISK
DT SKNTVYLQMN SLRAEDTAS Y Y CAKDLY
YGPTTQSAFNLWGQGT SVTVSS
>alpha chain sTCR comparator
MANQVEQ SPQ SLIILEGKNVTLQCNYTVSPF
SNLRWYKQDTGRGPVSLTILDYAINTK SNG
SEQ ID NO . : 70
RYTATLDADTKQS SLHITASQL SD SASYICV
VNRADGLYIPTFGRGT SLIVHPYIQKPDP AV
YQLRD SKS SDK SVCLF TDFD SQTNVS Q SKDS
DVYITDKCVLDMRSMDFKSNSAVAWSNKS
DFACANAFNNSIIPEDT
>beta chain sTCR comparator
MAIQMTQ SP S SLSASVGDRVTITCRASQDIR
sT CR
NYLNWYQQKPGKAPKLLIYYT SRLESGVP S
comparator RF S GS GS GTDYTLTIS SLQPEDFATYYCQQG
NTLPWTFGQGTKVEIKGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQPGG SLR
L S C AAS GY SF T GYTMNWVRQ APGKGLEWV
SEQ ID NO . : 71
ALINP YK GV S TYNQKFKDRF TIS VDK SKNT A
YLQMNSLRAEDTAVYYCARSGYYGDSDWY
FDVWGQGTLVTVS SGGGGSDVKVTQ SSRYL
VKRTGEKVFLECVQDAPL SKMFWYRQDPG
LGLRLIYF SYDVKLKEKGDIPEGY S V SREKK
ERF SLILESAS TNQT SMYLCA S S SDQNS GDP
YEQYFGPGTRLTVTEDLKNVFPPEVAVFEP S
EAEISHTQKATLVCLATGFYPDHVEL SWWV
89
CA 03240046 2024- 6- 4
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NGKEVHSGVCTDPQPLKEQPALNDSRYALS
SRLRVSATFWQDPRNHFRCQVQF YGL SEND
EWTQDRAKPVTQIVSAEAWGRAD
>CDR-16
EVQLVESGGGSAQAGGSLRVSCVASGRPFT
SEQ ID NO.: 72 CDR-16 KYAWGWFRQAPGK AREFVATITWDGGKTD
YADSVKGRFTISKDSAENSIYLQMNSLKPED
TASYYCAADRNYCVGHRCYVRPDDYDYVV
GQGTSVTVS SA
>CDR-17
EVQLVESGGGLVQPGGSLRLSCVASGRPFTK
SEQ ID NO.: 73 CDR-17 YAWGWFRQAPGKAREFVATITWDGGKTDY
AD S VKGRF TISKD SAKN SIYLQMN SLRAEDT
AVYYCAADRNYCVGIIRCYVRPDDYDYWG
QGTLVTVSS
>CDR-18 HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKD
TYIHAVVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDT
SEQ ID NO.: 74 AVYYC SRWGGDGFYAMDYWGQGTLVTVS
SASTKGPSVFPLAPS SKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQS SG
CDR-18 LYSLSSVVTVPSSSLGTQTYICNVNEIKPSNT
KVDKRVEPKSC
->CDR-18 LC
DIQMTQSPSSLSASVGDRVTITCRASQDVNT
SEQ ID NO.. 75 AVAWYQQKPGK APKLLIYS A SFLYSGVP SR
FSGSRSGTDFTLTISSLQPEDFATYYCQQHYT
TPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNAL
CA 03240046 2024- 6- 4
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Q SGNSQESVTEQD SKD S TY SL S STLTLSKAD
YEKHKVYACEVTHQGLSSPVTKSFNRGECG
GGGSQAVVTQEPSLTVSPGGTVTLTCRSSTG
AVTTSNYANWVQQKPGQAPRGLIGGTNKR
AP GVP ARF S G SLL GGKAAL T I S GAQPEDEAD
Y YCALW Y SNHW VFGGGTKLTVLGGGGGSG
GGGSGGGGSGGGGSEVQLVESGGGSVQPG
GSLRL S C AA S GF TF S T YAMNWVRQ AP GK GL
EWVGRIRSKYNNYATYYAD SVKGRF TISRD
DSKNTLYLQMNSLRAEDTAMYYCVRHGNF
GNSYVSWFAYWGQGTTVTVSS
SEQ ID NO 76 CD3 CDRH1 STYAMN
SEQ ID NO.. 77 CD3 CDRH2 RIRSKANNYATYYADSVKG
SEQ ID NO.. 78 CD3 CDRH3 HGNFGDSYVSWFAY
CD3
SEQ ID NO.: 79 GS STGAVTT SNYAN
CDRL1 a
CD3
SEQ ID NO.: 80 RS STGAVTT SNYAN
CDRL1 b
SEQ ID NO.: 81 CD3 CDRL2 GTNKRAP
SEQ ID NO.: 82 CD3 CDRL3 ALWYSNHWV
AVVTQEP SL TV SP GGTVTLTCRS STGAVTT S
NYANVVVQQKPGKSPRGLIGGTNKRAPGVP
SEQ ID NO.: 83 VL CD3 a
ARF SGSLLGGKAAL TIS GAQPED EAD YYC AL
WY SNHWVF GGGTKL TVLG
EVQLVESGGGSVQPGGSLRL S C AA S GF TF ST
YAMNWVRQAPGKGLEWVGRIRSKFNNYAT
SEQ ID NO.. 84 VH CD3 _a YYADSVKGRFTISRDDSKNTLYLQMNSLRA
EDTATYYCVRHGNFGDSYVSWFAYWGQGT
TVTVS S
91
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AVVTQEPSLTVSPGGTVTLTCGS STGAVTT S
NYANWVQQKPGK SPRGLIGGTNKRAPGVP
SEQ ID NO.: 85 VL CD3 b
ARF S GSLL GGKAAL TIS GAQPEDEADYYC AL
WYSNHWVFGGGTKLTVL
EVQLVESGGGSVQPGGSLRL SCAASGF TF ST
YAMNWVRQAPGKGLEWVGRIR SKANNYA
SEQ ID NO.: 86 VH CD3 b TYYADSVKGRFTISRDDSKNTLYLQMNSLR
AEDTATYYCVRHGNFGDSYVSWFAYWGQG
TTVTVSS
EXAMPLES
[0359] Intracellular tumor antigens presented as peptides on MEIC (pMHC) class
I
molecules are attractive targets for more tumor-selective immunotherapeutic
approaches with
promising data already emerging from clinical trials. pMHCs have been targeted
by TCR-
engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an
anti-CD3 fragment.
Naturally occurring cancer reactive TCRs have weak affinity and require
substantial affinity
enhancements for their cognate pMHC. However, the outcome of this process is
difficult to
predict and bears the risk for off-target cross reactivities in normal
tissues, which may lead to
severe adverse events in the clinic.
[0360] Here, we describe highly potent antigen binding proteins having a dual
pMHC T-
cell engager ("TCE") format with high specificity towards tumor-specific pMHCs
utilizing the
HLA-A*02.01 restricted MAGE-A4 epitope GVYDGREHTV (SEQ ID NO 1). A series of
monovalent and bivalent antibody constructs composed of anti-MAGE-A4 binding
arms, ranging
in affinities from 30 nM to 100 pM, were fused to an anti-CD3 Fab fragment
with lower affinity
compared to that commonly used for TCR-fusions. The different antibody
constructs were
evaluated for selective killing of MAGE-A4/HLA-A*02 positive human U2OS
osteosarcoma and
A375 melanoma cancer cells versus a panel of different MAGE-A4-negative/HLA-
A*02-positive
human cell lines. Bivalent bispecific antibody variants mediated at least a 7-
fold greater degree
of cancer cell killing and similarly increased T cell activation compared to
their monovalent
bispecific counterparts. IC50 values ranged as low as single digit picomolar,
while the overall
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cross reactivity against MAGE-A4-negative/HLA-A*02-positive cells was not
substantially
affected. These results prove that dual targeting of pMI-ICs on cancer cells
provides selective and
efficient T cell-mediated target cell killing and T cell activation, even at
very low levels of pMFIC
on the cell surface, highlighting the pivotal roles played by the affinity of
the individual arms,
valency, and epitope densities. The benefit of dual pMI-IC targeting was also
tested for other than
MAGE-A4fHLA-A*02 pMECs. T cell engagers specific for two distinct cancer-
derived p1V1HCs
unrelated to MAGE-A4 were tested in cytotoxicity assays in mono- and bivalent
formats. Alike
MAGE-A4 targeting TCEs, dual engagers showed improved cancer cell killing,
compared to their
monovalent counterparts. The MAGE-A4/HLA-A*02:01-targeting dual pMTIC TCE was
optimized for CD3 affinity and MAGE-A4/HLA-A*02:01 target affinity to achieve
high potency
while maintaining specificity by minimizing binding to similar and
physiologically relevant non-
MAGE-A4 peptides (Si, S16). We analyzed the optimized dual pMI-IC TCE for
potential off-
target effects by recognition of similar and physiologically relevant non-MAGE-
A4 peptides. T2
cells pulsed with similar peptides and co-cultured with PBMC effector cells
showed no significant
T cell activation or IFNg release in the presence of the dual pMFIC TCE in
comparison to MAGE-
A4 peptide-pulsed T2 cells. Finally, we compared the potency, cytokine
release, and specificity
of the dual plVIEIC TCE against a recombinant TCR fused to an anti-CD3 scFv, a
construct that is
currently in clinical development. Interestingly, the dual pMHC TCE resulted
in a 3-fold more
potent cancer cell killing while having significantly lower effect on cytokine
production. In
conclusion, pMFIC targeting with the dual pMI-IC TCEs described herein is an
attractive
alternative to soluble affinity-enhanced TCR-based cancer immunotherapies as
they facilitate
potent tumor targeting without the need for extensive affinity enhancements.
The dual pMTIC
TCEs provided herein show (i) selective and efficient T cell-mediated target
cell killing, (ii)
effective activation of T-cells and (iii) lower cytokine release than
comparator molecule. Dual
pMHC targeting with the antigen binding proteins provided herein is highly
potent while lower
cytokine release may avoid T cell exhaustion, thus providing the promise of
more effective and
durable anticancer responses.
Example 1 ¨ General method for production of monovalent and bivalent
pMHC-Targeting T Cell Engagers
[0361] Bispecific antigen binding proteins as described in the examples below
were
expressed by transient co-transfection in HEK293-6E cells. Cells were cultured
in suspension
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using polyethylenimine (PEI 401(D linear). HEK293-6E cells were seeded at 1.7
x 106 cells / mL
in Freestyle F17 medium supplemented with 2 mM L-Glutamine. DNA and PEI were
added
separately to 50 L, medium without supplement. Both fractions were mixed at
1:2.5 DNA:PEI
ratio, vortexed and rested for 15 minutes. Cells and DNA/PEI mixture were
combined (1 ittg
DNA/mL cells) and incubated at 37 C, 5% CO2, 80% RH. After 24 hours, cells
were
supplemented with Tryptone Ni at 25 1,IL per mL production volume. After 7
days, cells were
harvested by centrifugation and the supernatant was sterile filtered. The
antigen binding proteins
were purified by an affinity chromatography from the supernatant. Supernatant
was loaded on a
protein CH column (Thermo Fisher Scientific, 11494320005) equilibrated with 6
CV PBS (pH
7.4). After a washing step with the same buffer, protein was eluted from the
column by step elution
with 100 mM Citric acid (pH 3.0). Fractions with the desired antigen binding
protein were
immediately neutralized by 1 M Tris Buffer (pH 9.0) at 1:10 ratio. Size
exclusion chromatography
was performed as an additional purification step. Samples were run on the
Superdex 200 10/300
GL column with PBS (pH7.4) as a running buffer. Collected fractions were
analyzed by SE-HPLC
for monomer content and pooled accordingly. Final protein purity was assessed
by SDS-PAGE
and SE-HPLC.
Example 2 ¨ General methods for in vitro characterization of the bispecific
pMHC targeting T Cell Engagers
[0362] Affinity characterization of I-11,A-A2/MAGE-A4xCD3 bispecific
antibodies was
performed by surface plasmon resonance (SPR). All experiments were conducted
using a
BiacoreTM T200 Device (Cytiva). To determine the kinetic parameters of the
binding of the
bispecific antibodies to the HLA-A2/MAGE-A4 complex, a streptavidin chip
(SAHC30M,
XanTec) was coated according to the manufacturer's instructions with 500 RU
HLA-A*02:01 in
complex with the MAGE-A4 peptide. The resulting affinities presented herein
correspond to the
measurements performed with the respective monovalent antigen binding
proteins. To determine
the kinetic parameters of the bispecific antibodies to CD3, a HC3OM chip
(XanTec) was coated
according to the manufacturer's instructions with 400 RU of CD3 heterodimer
(Acro Biosystems).
Uncoated channels were used for referencing. Data fitting was performed using
a 1:1 Langmuir
model.
[0363] To determine the in vitro cytotoxicity of the bispecific antibodies the
Lactate
Dehydrogenase (LDH) release assay was performed. Briefly, target cells were co-
cultured with
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effector cells (i.e., PBMCs) at an E:T ratio of 10:1. Solutions of the
bispecific antibodies covering
a concentration range from 0.001 nM to 15 nM were added to the relevant wells.
Cytotoxicity was
quantified by colorimetric absorbance measurements of the amount of LDH
released from
damaged cells into the medium after 48 h. Cytokine release was determined
after 24 h.
Quantification of IL-2 and IFNg was performed using a respective cytokine
ELISA kit
(Invitrogen).
[0364] Thermal stability of the bispecific antibodies was measured using a
differential
scanning fluorimentry (DSF), as described in the Protein Thermal Shift manual
MAN4461806B
from Applied Biosystems (Thermo Fisher).
Example 3 ¨ Generation and characterization of various bispecific antibody
formats
[0365] To determine the most optimal format of the bispecific molecule,
rotation of the
VHFI MAGE-A4 binding moiety was performed on the N- and C-terminus of the
light chain, and
the C- and N- terminus of the heavy chain (formats 1-4 and respectively
compounds CDR1, CDR-
2, CDR-3 and CDR-4, Fig. 1) of a CD3 binding Fab. Monovalent bispecific T-cell
engagers were
tested for their affinity to MAGE-A4, as determined by SPR, in vitro potency,
as determined by
the LDH assay, and thermal stability, as determined by DSF. Results are
summarized in Table 5.
The respective T cell-mediated cytotoxicity results are shown in Figs. 2A-B.
Table 5 - Comparison of affinity, cytotoxicity, thermal stability, and
expression
yield of HLA-A2/MAGE-A4-specific monovalent bispecific antibodies in various
Fab-
VHH formats.
a 1-ILA-
U2OS cell Melting
Expressi
A 2/M A G
Compound Format killing, IC50 temperature on
yield
E-A4 KD
(nM) ( C)
(mg/L)
(nM)
CDR-1 Format #1 1.5 0.10 70.9
8.2
CDR-2 Format #2 2.6 0.18 71.3
8.2
CDR-3 Format #3 1.9 0.24 71.3
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CDR-4 Format #4 1.3 0.50 70.7
10.6
[0366] Comparative analysis of the various monovalent bispecific antibody
formats
revealed that positioning of the MAGE-A4 binding moiety on the CD3 binding Fab
impacted
neither the affinity to the MAGE-A4 antigen nor the thermal stability of the
construct. However,
N-terminal heavy chain fusion of the MAGE-A4 binding arm (i.e., format #4)
resulted in
reduction of the in vitro efficacy, likely due to steric hindrance of the CD3
binding arm. In
addition, a C-terminal fusion of the MAGE-A4 binding arm on the heavy chain of
the CD3
binding arm resulted in over a 4-fold increase in the expression yield.
[0367] It was hypothesized that a bivalent pMHC-targeting 1 cell engager could
mimic
the natural avidity of T cells through the binding of two pMHC molecules on
the surface of a
single tumor cell (without being bound to theory). Thus, a dual (i.e.,
bivalent) pMTIC-MAGE-A4-
targeting T cell engager was compared against a monovalent pMHC-MAGE-A4-
targeting T cell
engager. For the bivalent bispecific constructs, a C-terminal fusion of the
MAGE-A4 targeting
VEILI on the heavy chain in combination with N- or C-terminal fusion on the
light chain were
investigated (formats 5 and 6, compounds CDR-5 and CDR-6, respectively, Fig.
1). Comparison
of the two bivalent bispecific formats in cytotoxicity assays, thermal
stability and expression yield
was performed. Results are summarized in Table 6. The respective T cell-
mediated cytotoxicity
results are shown in Fig. 2C. Formats #5 and #6 showed excellent in vitro
efficacy, with IC50
values approximately 10-fold lower compared to the monovalent variants,
confirming the
superiority of the bivalent MAGE-A4 binding modus over the monovalent. Format
#6 showed
slightly higher thermal stability and better expression yield compared to
format #5.
Table 6 - Comparison of cytotoxicity, thermal stability and expression yield
of
HLA-A2/MAGE-A4-specific bivalent bispecific antibodies in various formats.
U2OS cell killing, Melting
Expression yield
Compound Format
IC50 (nM) temperature ( C)
(mg/L)
Format
CDR-5 0.01 68.8
32.8
#5
Form at
CDR-6 0.02 71.5
35.4
#6
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A direct comparison of the monovalent and the bivalent pMFIC T cell engager
(formats
#3 and #6) was performed (Fig. 2D). The bivalent pMFIC T cell engager
confirmed
superior cancer cell killing over its monovalent counterpart.
[0368] Finally, cytotoxicity in other cancer cell lines and the associated
cytokine release
profile were compared for the monovalent (compound CDR-3, format #3) and
bivalent
(compound CDR-6, format #6) pMHC T cell engagers. As shown in Fig. 3, percent
cancer cell
killing was measured in osteosarcoma (U2OS) and melanoma (A375) cells
incubated with a dual
pMHC-targeting T cell engager or a single pIVIHC-targeting T cell engager
comprising the same
MAGE-A4 and CD3-binding antibody fragments. The MAGE-A4 and HLA-A*02 positive
cell
line U2OS (osteosarcoma) was incubated with human PBMCs at an E:T ratio of
10:1 (Fig. 3A).
Similarly, MAGE-A4 and HLA-A*02 positive cell line A375 (melanoma) was
incubated with
human PBMCs at an E:T ratio of 10:1 (Fig. 3B). Cancer cell killing was
measured at various
concentrations of the two antigen binding proteins with an LDH release assay
after 48 hours. The
data shows a 10-fold increase in cancer cell killing potency with a dual pMTIC-
targeting T cell
engager compared to a single pMHC-targeting T cell engager. T cell activation
was determined
by quantification of CD69 and CD25 markers on the CD8 T cell population after
24h using flow
cytometry (Figs 3 C ¨ D), showing T cell activation on the U2OS (Fig. 3C) and
the A375 (Fig.
3D) cell line, respectively. The bivalent Fab-(VHH)2 format of the MAGE-A4
targeting TCE
shows superior cancer cell killing and T cell activation compared to its
monovalent counterpart.
Thus, bivalent targeting of antigen positive cancer cells greatly potentiates
activity of the pMHC-
targeting T-cell engagers. In this example, each antigen binding protein
utilized a low affinity
anti-CD3 Fab (see Example 4).
[0369] Further investigation of mono- and bivalent pMT1C targeting TCEs in
formats #3
and #6, where the pMHC binding moieties comprised scFvs was performed.
Compounds tested
were CDR-7 and CDR-8, respectively. Schematic representation of compound CDR-
8, a dual
engager with two pMHC-specific binding domains in a scFy format and a Fab
domain targeting
CD3 as a T cell recruiting domain is depicted in Fig. 4. Dual engager CDR-8
and its monovalent
counterpart CDR-7 were tested for efficacy in LDH assay. MAGE-A4-positive HLA-
A*02:01-
positive osteosarcoma cell line U2OS was incubated with human PBMCs at an E:T
ratio of 10:1.
Cancer cell killing was measured at various concentrations of the compounds
(Fig. 5). Again, data
showed superiority of the dual T cell engager over its monovalent counterpart
with about a 10-
fold increase in cancer cell killing potency.
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[0370] As further proof of concept, the benefit of bivalent targeting of
pIVIFICs was also
tested in other cancer cell lines and for other target pMI-1Cs. Two different
pM1-1C targeting T cell
engagers targeting two distinct - pMHC antigens, i.e., target A and B, were
generated in mono-
(i.e., Fab-scFv) and bivalent (i.e., Fab-(scFv)2) formats, and tested in
cytotoxicity assays. Briefly,
lung squamous cell carcinoma (expressing target A) and colorectal
adenocarcinoma (expressing
target B) cells were incubated with human PBMCs at an E:T ratio of 10:1 and
varying
concentrations of mono- and bivalent pMHC targeting TCEs. Cytotoxicity was
measured after 48
h incubation using the CellTiter-Glo Luminescent Cell Viability Assay
(Promega), according
to the manufacturer's instructions. Results are shown in Fig. 6. Alike MAGE-A4
targeting TCEs,
bivalent pMI-1C TCEs specific for other unrelated targets showed improved
cancer cell killing,
compared to their monovalent counterparts.
Example 4 - CD3 affinity of the bivalent ODIC-targeting T cell engager
influences the T cell-mediated cytotoxicity and the corresponding cytokine
release
[0371] Dual pMHC T cell engagers with MAGE-A4 arms comprising two identical
VHHs
(Fig. 7A) or scFvs (Fig. 7B) with low (54 nM), mid (11 nM) and high (1.2 nM)
CD3 affinity Fabs
were tested in the LDH assay on MAGE-A4-positive U2OS cells and MAGE-A4-
negative H441
cells. CDR-9, CDR-10 and CDR-11 comprised Fab-(V1-1H)2 compounds with low, mid
and high
affinity CD3 binding, respectively. CDR-12, CDR-13 and CDR-14 comprised Fab-
(scFv)2
compounds with low, mid and high affinity CD3 binding, respectively. Low
affinity CD3 binding
lead to lower potency, while high and mid affinity CD3 binding showed
increased cytotoxic
effects, correlating with the increasing CD3 affinity.
[0372] As shown in Fig. 8, cytokine release was detected in antigen-positive
osteosarcoma cells co-incubated with healthy donor PBMCs (E:T 10:1) and three
dual pMHC-
targeting T cell engagers in Fab-(VI-11-1)2 format, each with a different
level of binding affinity
for CD3, i.e., low (CDR-9, 54 nM), mid (CDR-10, 11 nM) and high (CDR-11, 1.2
nM). Cytokines
IL-2 and IFN gamma were measured at various concentrations of the three
antigen binding
proteins after a 24-hour incubation. The cytokines were measured using ELISA.
Collectively, the
results show that the level of cytokine release and potency can be tuned as
necessary by changing
the binding affinity of the anti-CD3 binding domain.
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Example 5 ¨ Potency of the bivalent pIVII1C TCE is strongly influenced by the
intrinsic affinity of the MAGE-A4 binding arms
[0373] Dual pMEIC-targeting T-cell engagers in Fab-(scFv)2 format comprising
low
(CDR-15) and high (CDR-8) affinity MAGE-A4 binders (KD of 41 nlVI and 0.1 nM,
respectively)
were evaluated for cell killing of MAGE-A4 positive U2OS cancer cells upon co-
incubation with
PBMCs (E:T 10:1). T cell-mediated cytotoxicity was determined by measuring LDH
release after
48h. The results as shown in Fig. 9 confirm that affinity enhancement of the
MAGE-A4 binding
arms mediates greater degree of cancer killing than the enhancement of the CD3
binding arm.
Example 6 - Dual pMHC TCE shows high selectivity compared to sTCR
comparator
[0374] Dual pM_HC TCE in Fab-(scFv)2 format (i.e., CDR-8) was analyzed for
potential
off-target effects by recognition of similar and physiologically relevant non-
MAGE-A4 peptides.
Applying in sitico analysis of peptide sequence similarity combined with mass
spectroscopy
analysis of eluted EILA peptides, peptide databases and alanine scanning, the
specificity of
MAGE-A4/MHC-targeting antibodies was previously evaluated (KA1VIAR PELED et
al., 2015).
The identified similar peptides (Si, S16) with confirmed human tissue
expression were separately
loaded on the TAP-deficient T2 cells, which express empty HLA-A*02.01
molecules on the
surface, for specificity assessment.
[0375] TAP-deficient T2 cells were pulsed with HLA-A*02.01 -restricted
peptides
(MAGE-A4 or similar control peptides, deemed to be presented in relevant human
tissues, Si
(GLADGRTHTV, SEQ ID NO.: 89) and S16 (GLYDGPVHEV, SEQ ID NO.: 90)) and co-
incubated with PBMCs (E:T 5:1) and 0.1 n1\4 of the dual pMHC-targeting TCE
comprising the
high affinity MAGE-A4 scFvs and mid affinity CD3 Fabs or an in-house produced
clinical stage
comparator molecule (sTCRxCD3). The Comparator is composed of a soluble TCR
with binding
specificity for the same pMHC-MAGE-A4 antigen with an 87 pM KD, linked to an
anti-CD3 scFy
with a 1 nM KD and therewith similar to the clinical stage IMC-C103C compound.
The
comparator is monovalent for the target pMHC and CD3, while the dual engager
is bivalent for
the target pMFIC and monovalent for CD3. The comparator molecule and the dual
engager are
schematically depicted in Fig. 10.
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[0376] T cell activation was determined by quantification of CD25 markers on
the CD8
T cell population after 24h using flow cytometry, see Fig. 11A. T2 cells were
treated as described
above, incubated with 0.1 nM dual pMHC-targeting T cell engager comprising
high MAGE-A4
and mid CD3 affinity. Cytokine release was determined by quantification of IFN-
gamma in the
cell supernatants after 24h using ELISA (results depicted in Fig. 11B). The
results show that the
dual pMTIC-targeting T cell engager (with picomolar affinity for MAGE-A4)
elicits considerably
lower T cell functional responses for the Si and S16 off-target peptides than
for the MAGE-A4
target peptide. Therefore, the bivalent targeting of MAGE-A4 does not
compromise selectivity of
the bispecific molecule since the T2 cells pulsed with similar physiologically
relevant peptides
and co-cultured with PBMC effector cells showed no significant T cell
activation or 1FNg release
in the presence of the dual pMHC TCE in comparison to MAGE-A4 peptide-pulsed
T2 cells.
Example 7 ¨ Bivalent pMHC TCE demonstrates limited cross-reactivity
towards antigen-negative cells in vitro
[0377] MAGE-A4 negative/HLA-A*02:01 positive cells (SK-MEL-30, NCI-H441,
MDA-MB-231, PANC-1) were co-incubated with PBMCs (E:T 10:1) and either dual
pMHC-
targeting T cell engager in Fab-(scFv)2 format with picomolar MAGE-A4-
targeting scFvs and a
CD3-targeting Fab having mid CD3 affinity (i.e., CDR-8) or an in-house
produced clinical stage
comparator molecule as described in example 6. T cell-mediated cytotoxicity
was determined by
measuring LDH release after 48h. Results are shown in Fig. 12. Accordingly,
dual pMHC-
targeting T cell engager induces comparable or less cytotoxicity of MAGE-A4
negative/HLA-
A*02:01 positive cells than sTCRxCD3 comparator.
Example 8 - Dual pMHC T cell engager shows high anti-tumor cytotoxicity
profile with limited cytokine release
[0378] As shown in Fig. 13, percent cancer cell killing in osteosarcoma cells
and
melanoma cells incubated with the dual pMHC-targeting T cell engager in Fab-
(scFv)2 format
(i.e., CDR-8) or comparator as described in example 6 was measured. MAGE-A4 &
IALA-A*02
positive cell lines A375 (melanoma) and U2OS (osteosarcoma) were incubated
with human
PBMCs at an E:T ratio of 10:1 for 48 h. Cancer cell killing was measured at
various concentrations
of the two antigen binding proteins. The data shows that the dual pMHC-
targeting TCE more
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potently mediated killing of both cancer cell lines compared to Comparator.
This was true even
in the melanoma cell line, which only expresses a low copy number of the
target pMEIC-MAGE-
A4 antigen (about 35 copies per cell).
[0379] As shown in Fig. 14, cytokine release in osteosarcoma cells and
melanoma cells
incubated with the dual pMHC-targeting TCE (i.e., CDR-8) or Comparator was
measured.
Cytokine release was determined by quantification of IFN-gamma and IL-2 in the
cell
supernatants after 20h using ELISA. MAGE-A4 & HLA-A*02 positive cell lines
A375
(melanoma) and U2OS (osteosarcoma) were incubated with human PBMCs at an E:T
ratio of
10:1. Cytokines IL-2 and IFN gamma were measured at various concentrations of
the two antigen
binding proteins. The data shows that the dual engager induced lower levels of
the two pro-
inflammatory cytokines, indicating a lower potential for inducing a cytokine
storm syndrome.
[0380] Live cell imaging of MAGE-A4 positive NCI-H1703 lung squamous carcinoma
cells co-cultured with human PBMCs in presence of a dual pMI-IC TCE in Fab-
(scFv)2 format
(i.e., CDR-8) with specificity for MAGE-A4/HLA-A*02:01 was performed. Lung
cancer cells
were stained with Cytolight Rapid Red; cell death was revealed with Cytotox
Green. As shown
in Figs. 15 A and B, the dual pMHC-targeting TCE elicits highly efficient anti-
tumor responses.
Example 9 ¨ Reduced Anti-Drug Antibodies (ADAs) With pMHC-Targeting
T Cell Engagers
[0381] ADAs may affect the risk profile and efficacy of a biological drug. If
neutralizing,
they may block the drug's ability to bind to its target. It is therefore a
regulatory requirement to
test biologic drugs for the binding of anti-drug antibodies and their
neutralizing potential. In
addition, if the pre-existing Abs recognize the C-terminally located scFvs or
sdAbs, clustering of
T Cell engagers via binding to the pre-existing antibodies may occur. Such
phenomenon could
lead to generation of pre-existing ADA:bispecific Ab complexes with clustered
free T cell
engaging moieties. The presence of such complexes comprising multiple free CD3
binding arms
could lead to avidity-driven T cell activation in the absence of cancer cells,
which could in turn
cause a cytokine release syndrome. The monovalent pMFIC-targeting T cell
engager was tested
for its ability to evade ADA binding compared to sTCR comparator.
[0382] As shown in Fig. 16, pre-existing ADAs were quantified for the
comparator, as
described above, and the de-immunized sdAb compound CDR-16. Comparator and the
de-
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immunized sdAb were evaluated with serum samples from 10 healthy naïve
Caucasian human
donors. Pre-existing ADAs were detected by ELISA. The data shows that the de-
immunized
sdAb was not targeted by ADAs, while Comparator was bound by ADAs.
[0383] In an effort to reduce ADA engagement with the dual OM-IC-targeting T
cell
engager format, amino acid modifications were generated in a single domain
antibody format
(sdAb) and an scFy format. As shown in Fig. 17, binding to pre-existing ADAs
was quantified
in humanized sdAb compound CDR-17 with selected modifications. "+A-
corresponds to the
addition of an alanine on C-terminus. "-S" corresponds to the deletion of a
serine at position 113,
according to Kabat numbering. "-SS" corresponds to the deletion of a serine at
position 112 and
113, according to Kabat numbering. "SSS" corresponds to the substitution of
hydrophobic amino
acids at Kabat positions 11, 89, and 108 to serine amino acids. The triple
serine substitution
-SSS" is further described in W02009/155725, incorporated herein by reference.
The ADA
response was measured with an ELISA over different sample serum
concentrations. The data
demonstrates that the inclusion of any one or more of the above modifications
reduces binding to
ADAs. The combination of SSS and -SS modifications or SSS, -SS, and A
modifications reduced
binding to ADA the most.
[0384] As shown in Fig. 18, binding to pre-existing ADAs was quantified for
Fab-scFy
antigen binding proteins based on compound CDR-18 with selected modifications
on the scFv.
"+A" corresponds to the addition of an alanine. "-S" corresponds to the
deletion of a serine at
position 113, according to Kabat numbering. "-SS" corresponds to the deletion
of a serine at
position 112 and 113, according to Kabat numbering. "SSS" corresponds to the
substitution of
hydrophobic amino acids at Kabat positions 11, 89, and 108 to serine amino
acids. The ADA
response was measured with an ELISA over different sample serum
concentrations. The data
demonstrates that the inclusion of any one or more of the above modifications
reduces binding to
ADAs.
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